Sequencing method for rapid identification and processing of cognate nucleotide pairs

ABSTRACT

Provided are methods and systems for reducing the time needed for sequencing nucleic acids. The approach relies on detecting formation of nucleotide-specific ternary complexes comprising a polymerase (e.g., a DNA polymerizing enzyme), a primed template nucleic acid molecule, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide, as well as the subsequent next correct nucleotide from a cycle of examining four different nucleotides without requiring chemical incorporation of any nucleotide into the primer.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/375,389, filed Aug. 15, 2016. The entire disclosure of this earlierapplication is hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates generally to the field of biotechnology.More specifically, the invention concerns nucleic acid sequencingtechnology.

BACKGROUND

Simplifying large-scale sequencing projects, such as genome-scaleprojects, depends on rapid acquisition of DNA sequence data.Unfortunately, nucleic acid sequencing procedures typically involvetime-consuming cycling of reagents, enzymatic reactions that must go tocompletion, and potentially long periods of acquiring measurement data.Of course, all of this is a legacy of the early nucleic acid sequencingparadigms that relied on processing one nucleotide position at a time.

Alternative methods aimed at identifying short stretches of sequence atone time (e.g., using sequencing-by-hybridization) have not found wideacceptance for determining unknown sequences. Instead, these techniquesgenerally are applied to investigating subtle changes in sequences thatalready are known. The same is true for techniques that are based on“melting curve analysis,” where the interrogation sequences are commonlyno larger than about twenty nucleotides in length.

The techniques described herein provide an approach that advantageouslyspeeds acquisition of nucleic acid sequence data and provide otheradvantages as well.

SUMMARY OF THE DISCLOSURE

In one aspect, the disclosure relates to a method of determining theidentity of first and second correct nucleotides respectively includingbases complementary to the next two bases of a template strandimmediately downstream of a primer of a primed template nucleic acidmolecule. The method includes the step of (a) conducting a plurality ofcycles of contacting a first primed template nucleic acid molecule witha reaction mixture that includes a polymerase and, for each cycle, adifferent test nucleotide, and removing any polymerase and nucleotidethat may have bound to the first primed template nucleic acid molecule.The polymerase does not incorporate any of the different testnucleotides into the primer of the first primed template nucleic acidmolecule during any of the plurality of cycles. There also is the stepof (b) measuring, for each of the cycles in step (a), signals indicatingbinding of the first primed template nucleic acid molecule to thepolymerase and one of the different test nucleotides to identify a testnucleotide associated with the highest magnitude of ternary complexformation, and a test nucleotide associated with the second-highestmagnitude of ternary complex formation. There also is the step of (c)determining the identities of the first correct nucleotide downstream ofthe primer, and the second correct nucleotide downstream of the primerusing the measured binding signals from step (b). According to onegenerally preferred embodiment, the primer of the first primed templatenucleic acid molecule is an extendable primer that does not include areversible terminator moiety attached to its 3′ terminal nucleotide.When this is the case, the reaction mixture can include either adivalent non-catalytic metal ion that inhibits polymerase-mediatedincorporation, or a trivalent non-catalytic metal ion that inhibitspolymerase-mediated incorporation. Alternatively, the plurality ofcycles in step (a) can include four cycles. According to anotheralternative, step (c) can involve determining that the test nucleotideassociated with the highest magnitude of ternary complex formation isthe first correct nucleotide downstream of the primer, and that the testnucleotide associated with the second-highest magnitude of ternarycomplex formation is the second correct nucleotide downstream of theprimer. According to still a different alternative, the method furtherincludes the step of (d) incorporating a reversible terminatornucleotide including a reversible terminator moiety into the firstprimed template nucleic acid molecule to produce a first blocked primedtemplate nucleic acid molecule. More preferably, there is the furtherstep of (e) removing the reversible terminator moiety from the firstblocked primed template nucleic acid molecule to produce a firstde-blocked primed template nucleic acid molecule. Still more preferably,the method further involves repeating steps (a)-(c) using the firstde-blocked primed template nucleic acid molecule in place of the firstprimed template nucleic acid molecule. Alternatively, the method furtherincludes the steps of: (f) incorporating a reversible terminatornucleotide including a reversible terminator moiety into the firstde-blocked primed template nucleic acid molecule to produce a secondblocked primed template nucleic acid molecule; and (g) removing thereversible terminator moiety from the second blocked primed templatenucleic acid molecule to produce a second de-blocked primed templatenucleic acid molecule. More preferably, the method further involvesrepeating steps (a)-(c) using the second de-blocked primed templatenucleic acid molecule in place of the first primed template nucleic acidmolecule. More preferably, step (c) occurs after all of steps (a)-(b)and (d)-(g) have been performed. Optionally, none of the different testnucleotides includes an exogenous fluorescent label. When this is thecase, each of the different test nucleotides can be a different nativenucleotide. Optionally, the polymerase does not include an exogenousfluorescent label, and step (b) does not include measuring anyfluorescent signal from the polymerase. According to another generallypreferred embodiment, the first primed template nucleic acid molecule isa first blocked primed template nucleic acid molecule, and the primer ofthe first blocked primed template nucleic acid molecule includes areversible terminator moiety that blocks phosphodiester bond formation.When this is the case, the reaction mixture can include a catalyticmetal ion. Alternatively, the plurality of cycles in step (a) caninclude four cycles. According to another alternative, step (c) involvesdetermining that the test nucleotide associated with the highestmagnitude of ternary complex formation is the first correct nucleotidedownstream of the primer, and that the test nucleotide associated withthe second-highest magnitude of ternary complex formation is the secondcorrect nucleotide downstream of the primer. According to still anotheralternative, the method further includes the steps of: (d) removing thereversible terminator moiety from the primer of the first blocked primedtemplate nucleic acid molecule to produce a first de-blocked primer; and(e) incorporating a reversible terminator nucleotide including areversible terminator moiety into the first de-blocked primer to producea second blocked primed template nucleic acid molecule. More preferably,steps (a)-(c) are repeated using the second blocked primed templatenucleic acid molecule in place of the first blocked primed templatenucleic acid molecule. Alternatively, the method further includes, afterstep (e), the steps of: (f) removing the reversible terminator moietyfrom the second blocked primed template nucleic acid molecule to producea second de-blocked primer; and (g) incorporating a reversibleterminator nucleotide including a reversible terminator moiety into thesecond de-blocked primer to produce a third blocked primed templatenucleic acid molecule. More preferably, steps (a)-(c) can be repeatedusing the third blocked primed template nucleic acid molecule in placeof the first blocked primed template nucleic acid molecule. Still morepreferably, step (c) occurs after all of steps (a)-(b) and (d)-(g) havebeen performed. Optionally, in any of the embodiments none of thedifferent test nucleotides includes an exogenous fluorescent label. Whenthis is the case, each of the different test nucleotides can be adifferent native nucleotide. Optionally, in any of the embodiments thepolymerase does not include an exogenous fluorescent label, and step (b)does not include measuring any fluorescent signal from the polymerase.According to still yet another embodiment, when the first primedtemplate nucleic acid molecule is a first blocked primed templatenucleic acid molecule, and when the primer of the first blocked primedtemplate nucleic acid molecule includes a reversible terminator moietythat blocks phosphodiester bond formation, the reversible terminatormoiety that blocks phosphodiester bond formation can be a 3′-ONH₂moiety.

In another aspect, the disclosure relates to a method of identifying anucleotide that includes a base complementary to the next base of atemplate strand immediately downstream of the primer of a primedtemplate nucleic acid molecule. The method includes the step of (a)contacting the primed template nucleic acid molecule with a firstreaction mixture that includes a polymerase, a reversible terminatornucleotide that includes a reversible terminator moiety, and a catalyticmetal ion. The reversible terminator nucleotide can incorporate at the3′-end of the primer of the primed template nucleic acid molecule toproduce a reversibly blocked primed template nucleic acid molecule.There also is the step of (b) contacting the reversibly blocked primedtemplate nucleic acid molecule with a second reaction mixture thatincludes a polymerase, at least one nucleotide molecule, and a catalyticmetal ion. A ternary complex forms if one of said at least onenucleotide molecule includes the base complementary to the next base ofthe template strand. There also is the step of (c) monitoringinteraction of the polymerase from the second reaction mixture and thereversibly blocked primed template nucleic acid molecule to detect anyof the ternary complex that may have formed. There also is the step of(d) determining either that: one of the at least one nucleotide moleculeof the second reaction mixture is the next correct nucleotide if theternary complex is detected in step (c), or none of said at least onenucleotide molecule of the second reaction mixture is the next correctnucleotide if the ternary complex is not detected in step (c). Accordingto one generally preferred embodiment, the at least one nucleotidemolecule includes a plurality of nucleotide molecules, where the ternarycomplex forms if one of the plurality of nucleotide molecules includesthe base complementary to the next base of the template strand, andwhere step (d) includes determining either that: one of the plurality ofnucleotide molecules of the second reaction mixture is the next correctnucleotide if the ternary complex is detected in step (c), or none ofthe plurality of nucleotide molecules of the second reaction mixture isthe next correct nucleotide if the ternary complex is not detected instep (c). Optionally, the method further includes the steps of: (e)removing the reversible terminator moiety from the reversibly blockedprimed template nucleic acid molecule to produce an unblocked primedtemplate nucleic acid molecule; and (f) repeating steps (a)-(d) usingthe unblocked primed template nucleic acid molecule in place of theprimed template nucleic acid molecule. More preferably, the at least onenucleotide molecule in step (b) is only a single nucleotide molecule,the ternary complex forms if the single nucleotide molecule includes thebase complementary to the next base of the template strand, and step (d)includes determining either that: the single nucleotide molecule of thesecond reaction mixture is the next correct nucleotide if the ternarycomplex is detected in step (c), or the single nucleotide molecule ofthe second reaction mixture is not the next correct nucleotide if theternary complex is not detected in step (c). Optionally, in any of theembodiments the primed template nucleic acid molecule is immobilized toa solid support, and steps (a) and (b) include flowing the reactionmixtures over the immobilized primed template nucleic acid molecule.Optionally, in any of the embodiments the catalytic metal ion ismagnesium ion or manganese ion. Optionally, in any of the embodimentsthe reversible terminator moiety includes a chemical moiety attached atthe 3′ position of the terminal nucleotide of the primer. When this isthe case, the chemical moiety includes a 3′-ONH₂ moiety. Optionally, inany of the embodiments step (c) can involve monitoring continuously.Optionally, the polymerase of the second reaction mixture includes anexogenous label. When this is the case, the exogenous label can includea fluorescent label. More preferably, the fluorescent label of thepolymerase of the second reaction mixture is not a conformationallysensitive fluorescent label that changes optical properties uponinteraction with a nucleotide. Still more preferably, the polymerases ofthe first and second reaction mixtures are different DNA polymerases.Optionally, step (d) involves removing with a chemical reagent.Optionally, each of the at least one nucleotide molecules is a nativenucleotide molecule that does not include an exogenous fluorescentlabel.

In another aspect, the disclosure relates to a method of determining theidentity of first and second correct nucleotides respectively includingbases complementary to the next two bases of a template strandimmediately downstream of a primer in a primed template nucleic acidmolecule. The method includes the step of (a) conducting a plurality ofcycles of contacting a first primed template nucleic acid molecule witha reaction mixture including a polymerase and, for each cycle, adifferent pair of test nucleotides, and removing any polymerase andnucleotide that may have bound to the first primed template nucleic acidmolecule, where the polymerase does not incorporate any of the differenttest nucleotides into the primer of the first primed template nucleicacid molecule during any of the four cycles. There also is the step of(b) measuring, for each of the cycles in step (a), signals indicatingbinding of the first primed template nucleic acid molecule to thepolymerase and at least one of the test nucleotides among the differentpairs of test nucleotides to identify a test nucleotide associated withthe highest magnitude of ternary complex formation, and a testnucleotide associated with the second-highest magnitude of ternarycomplex formation. There also is the step of (c) determining theidentities of the first correct nucleotide downstream of the primer, andthe second correct nucleotide downstream of the primer using themeasured binding signals from step (c). According to one generallypreferred embodiment, the plurality of cycles in step (a) includes atleast four cycles. According to a different generally preferredembodiment, step (c) can involve determining that the test nucleotideassociated with the highest magnitude of ternary complex formation isthe first correct nucleotide downstream of the primer, and the testnucleotide associated with the second-highest magnitude of ternarycomplex formation is the second correct nucleotide downstream of theprimer. According to still a different generally preferred embodiment,conducting the plurality of cycles in step (a) can involve conductingonly four cycles, and the different pairs of test nucleotides of thereaction mixture of step (a) can be any of: (i) dATP and dGTP, dATP anddCTP, dGTP and dTTP, dCTP and dTTP; (ii) dATP and dGTP, dATP and dTTP,dGTP and dCTP, dCTP and dTTP; and (iii) dATP and dCTP, dATP and dTTP,dGTP and dCTP, dGTP and dTTP. According to still a different generallypreferred embodiment, conducting the plurality of cycles in step (a) caninvolve conducting six cycles, and the different pairs of testnucleotides of the reaction mixture can be: dATP and dGTP, dATP anddCTP, dATP and dTTP, dGTP and dCTP, dGTP and dTTP, and dCTP and dTTP.According to still yet a different generally preferred embodiment, themethod further includes the step of: (d) incorporating a reversibleterminator nucleotide including a reversible terminator moiety into thefirst primed template nucleic acid molecule to produce a first blockedprimed template nucleic acid molecule. When this is the case, the methodmay further include the step of: (e) removing the reversible terminatormoiety from the first blocked primed template nucleic acid molecule toproduce a first de-blocked primed template nucleic acid molecule. Morepreferably, the method further involves repeating steps (a)-(c) usingthe first de-blocked primed template nucleic acid molecule in place ofthe first primed template nucleic acid molecule. Alternatively, themethod further includes the steps of: (f) incorporating a reversibleterminator nucleotide including a reversible terminator moiety into thefirst de-blocked primed template nucleic acid molecule to produce asecond blocked primed template nucleic acid molecule; and (g) removingthe reversible terminator moiety from the second blocked primed templatenucleic acid molecule to produce a second de-blocked primed templatenucleic acid molecule. More preferably, the method further involvesrepeating steps (a)-(c) using the second de-blocked primed templatenucleic acid molecule in place of the first primed template nucleic acidmolecule. Optionally, in any of the embodiments that include step (d),step (c) occurs after all of the other steps have been performed.Optionally, in any of the embodiments none of the different testnucleotides includes an exogenous fluorescent label. Optionally, in anyof the embodiments each of the different test nucleotides is a differentnative nucleotide. Optionally, in any of the embodiments the polymerasedoes not include an exogenous fluorescent label, and step (b) does notinclude measuring any fluorescent signal produced by the polymerase.Optionally, in any of the embodiments the reaction mixture includes anon-catalytic metal ion that inhibits polymerase-mediated incorporation.Optionally, in any of the embodiments the reaction mixture includes acatalytic metal ion.

In another aspect, the disclosure relates to a method of determining theidentity of first and second correct nucleotides respectively includingbases complementary to the next two bases of a template strand in ablocked primed template nucleic acid molecule. The method includes thesteps of: (a) conducting a plurality of cycles of contacting a firstblocked primed template nucleic acid molecule with a reaction mixtureincluding a polymerase and, for each cycle, a different pair of testnucleotides, and removing any polymerase and nucleotide that may havebound to the first blocked primed template nucleic acid molecule, wherethe first blocked primed template nucleic acid molecule includes aprimer with a reversible terminator moiety that blocks phosphodiesterbond formation. There also is the step of (b) measuring, for each of thecycles in step (a), signals indicating binding of the first blockedprimed template nucleic acid molecule to the polymerase and at least oneof the test nucleotides among the different pairs of test nucleotides toidentify a test nucleotide associated with the highest magnitude ofternary complex formation, and a test nucleotide associated with thesecond-highest magnitude of ternary complex formation. There also is thestep of (c) determining the identities of the first correct nucleotidedownstream of the primer, and the second correct nucleotide downstreamof the primer using the measured binding signals from step (c).According to one generally preferred embodiment, the plurality of cyclesin step (a) involves at least four cycles. According to a differentgenerally preferred embodiment, step (c) involves determining that thetest nucleotide associated with the highest magnitude of ternary complexformation is the first correct nucleotide downstream of the primer, andthat the test nucleotide associated with the second-highest magnitude ofternary complex formation is the second correct nucleotide downstream ofthe primer. According to still a different generally preferredembodiment, conducting the plurality of cycles in step (a) can involveconducting only four cycles, and the different pairs of test nucleotidesof the reaction mixture of step (a) can be any of: (i) dATP and dGTP,dATP and dCTP, dGTP and dTTP, dCTP and dTTP; (ii) dATP and dGTP, dATPand dTTP, dGTP and dCTP, dCTP and dTTP; and (iii) dATP and dCTP, dATPand dTTP, dGTP and dCTP, dGTP and dTTP. According to yet a differentgenerally preferred embodiment, conducting the plurality of cycles instep (a) can involve conducting six cycles, and the different pairs oftest nucleotides of the reaction mixture can be: dATP and dGTP, dATP anddCTP, dATP and dTTP, dGTP and dCTP, dGTP and dTTP, and dCTP and dTTP.According to still yet a different generally preferred embodiment, themethod can further include the steps of: (d) removing the reversibleterminator moiety from the primer of the first blocked primed templatenucleic acid molecule to produce a first de-blocked primer; and (e)incorporating a reversible terminator nucleotide including a reversibleterminator moiety into the first de-blocked primer to produce a secondblocked primed template nucleic acid molecule. When this is the case,steps (a)-(c) can be repeated using the second blocked primed templatenucleic acid molecule in place of the first blocked primed templatenucleic acid molecule. More preferably, conducting the plurality ofcycles in step (a) can involve conducting only four cycles, and thedifferent pairs of test nucleotides of the reaction mixture of step (a)can be any of: (i) dATP and dGTP, dATP and dCTP, dGTP and dTTP, dCTP anddTTP; (ii) dATP and dGTP, dATP and dTTP, dGTP and dCTP, dCTP and dTTP;and (iii) dATP and dCTP, dATP and dTTP, dGTP and dCTP, dGTP and dTTP.Still more preferably, conducting the plurality of cycles in step (a)can involve conducting six cycles, and the different pairs of testnucleotides of the reaction mixture can be: dATP and dGTP, dATP anddCTP, dATP and dTTP, dGTP and dCTP, dGTP and dTTP, and dCTP and dTTP. Inaccordance with embodiments that include steps (d) and (e), the methodfurther includes, after step (e), the steps of: (f) removing thereversible terminator moiety from the second blocked primed templatenucleic acid molecule to produce a second de-blocked primer; and (g)incorporating a reversible terminator nucleotide including a reversibleterminator moiety into the second de-blocked primer to produce a thirdblocked primed template nucleic acid molecule. More preferably, steps(a)-(c) can be repeated using the third blocked primed template nucleicacid molecule in place of the first blocked primed template nucleic acidmolecule. Still more preferably, conducting the plurality of cycles instep (a) involves conducting only four cycles, and where the differentpairs of test nucleotides of the reaction mixture of step (a) are anyof: (i) dATP and dGTP, dATP and dCTP, dGTP and dTTP, dCTP and dTTP; (ii)dATP and dGTP, dATP and dTTP, dGTP and dCTP, dCTP and dTTP; and (iii)dATP and dCTP, dATP and dTTP, dGTP and dCTP, dGTP and dTTP.Alternatively, conducting the plurality of cycles in step (a) involvesconducting six cycles, and the different pairs of test nucleotides ofthe reaction mixture can be: dATP and dGTP, dATP and dCTP, dATP anddTTP, dGTP and dCTP, dGTP and dTTP, and dCTP and dTTP. Optionally, inany of the embodiments step (c) occurs after all of the other steps havebeen performed. Optionally, in any of the embodiments none of thedifferent test nucleotides includes an exogenous fluorescent label.Optionally, in any of the embodiments each of the different testnucleotides is a different native nucleotide. Optionally, in any of theembodiments the polymerase does not include an exogenous fluorescentlabel, and step (b) does not involve measuring any fluorescent signalfrom the polymerase. Optionally, in any of the embodiments the reactionmixture includes a non-catalytic metal ion that inhibitspolymerase-mediated incorporation. Optionally, in any of the embodimentsthe reaction mixture includes a catalytic metal ion.

In another aspect, the disclosure relates to a method of determining theidentity of first and second correct nucleotides respectively includingbases complementary to the next two bases of a template strandimmediately downstream of a primer in a primed template nucleic acidmolecule. The method includes the step of (a) conducting a plurality ofcycles of contacting a first primed template nucleic acid molecule witha reaction mixture that includes a polymerase and, for each cycle, adifferent test nucleotide, and removing any polymerase and nucleotidethat may have bound to the first primed template nucleic acid molecule.The polymerase does not incorporate any of the different testnucleotides into the primer of the first primed template nucleic acidmolecule during any of the plurality of cycles. There also is the stepof (b) measuring, for each of the cycles in step (a), signals indicatingbinding of the first primed template nucleic acid molecule to thepolymerase and one of the different test nucleotides to identify a testnucleotide associated with the highest magnitude of ternary complexformation, and a test nucleotide associated with the second-highestmagnitude of ternary complex formation. There also is the step of (c)determining the identities of the first correct nucleotide downstream ofthe primer, and the second correct nucleotide downstream of the primerusing the measured binding signals from step (b). According to onegenerally preferred embodiment, the plurality of cycles in step (a)includes four cycles. According to a different generally preferredembodiment, step (c) can involve determining that the test nucleotideassociated with the highest magnitude of ternary complex formation isthe first correct nucleotide downstream of the primer, and that the testnucleotide associated with the second-highest magnitude of ternarycomplex formation is the second correct nucleotide downstream of theprimer. According to still a different generally preferred embodiment,the method further includes the step of (d) incorporating a reversibleterminator nucleotide including a reversible terminator moiety into thefirst primed template nucleic acid molecule to produce a first blockedprimed template nucleic acid molecule. When this is the case, the methodcan further include the step of (e) removing the reversible terminatormoiety from the first blocked primed template nucleic acid molecule toproduce a first de-blocked primed template nucleic acid molecule. Morepreferably, the method can further involve repeating steps (a)-(c) usingthe first de-blocked primed template nucleic acid molecule in place ofthe first primed template nucleic acid molecule. Still more preferably,the method further includes the steps of: (f) incorporating a reversibleterminator nucleotide including a reversible terminator moiety into thefirst de-blocked primed template nucleic acid molecule to produce asecond blocked primed template nucleic acid molecule; and (g) removingthe reversible terminator moiety from the second blocked primed templatenucleic acid molecule to produce a second de-blocked primed templatenucleic acid molecule. Yet still more preferably, the method can furtherinvolve repeating steps (a)-(c) using the second de-blocked primedtemplate nucleic acid molecule in place of the first primed templatenucleic acid molecule. When this is the case, step (c) can occur afterall of the other steps have been performed. Optionally, in any of theembodiments none of the different test nucleotides includes an exogenousfluorescent label. More preferably, each of the different testnucleotides is a different native nucleotide. Optionally, in any of theembodiments the polymerase does not include an exogenous fluorescentlabel, and step (b) does not include measuring any fluorescent signalfrom the polymerase. Optionally, in any of the embodiments the reactionmixture includes a non-catalytic metal ion that inhibitspolymerase-mediated incorporation. Optionally, in any of the embodimentsthe reaction mixture includes a catalytic metal ion.

In another aspect, the disclosure relates to method of determining theidentity of first and second correct nucleotides respectively includingbases complementary to the next two bases of a template strand in ablocked primed template nucleic acid molecule. The method includes thestep of (a) conducting a plurality of cycles of contacting a firstblocked primed template nucleic acid molecule with a reaction mixtureincluding a polymerase and, for each cycle, a different test nucleotide,and removing any polymerase and nucleotide that may have bound to thefirst blocked primed template nucleic acid molecule, where the firstblocked primed template nucleic acid molecule includes a primer with areversible terminator moiety that blocks phosphodiester bond formation.There also is the step of (b) measuring, for each of the cycles in step(a), signals indicating binding of the first blocked primed templatenucleic acid molecule to the polymerase and one of the different testnucleotides to identify a test nucleotide associated with the highestmagnitude of ternary complex formation, and a test nucleotide associatedwith the second-highest magnitude of ternary complex formation. Therealso is the step of (c) determining the identities of the first correctnucleotide downstream of the primer, and the second correct nucleotidedownstream of the primer using the measured binding signals from step(c). According to one generally preferred embodiment, the plurality ofcycles in step (a) includes four cycles. According to a differentgenerally preferred embodiment, step (c) can involve determining thatthe test nucleotide associated with the highest magnitude of ternarycomplex formation is the first correct nucleotide downstream of theprimer, and that the test nucleotide associated with the second-highestmagnitude of ternary complex formation is the second correct nucleotidedownstream of the primer. According to still a different generallypreferred embodiment, the method further includes the steps of: (d)removing the reversible terminator moiety from the primer of the firstblocked primed template nucleic acid molecule to produce a firstde-blocked primer; and (e) incorporating a reversible terminatornucleotide including a reversible terminator moiety into the firstde-blocked primer to produce a second blocked primed template nucleicacid molecule. When this is the case, steps (a)-(c) can be repeatedusing the second blocked primed template nucleic acid molecule in placeof the first blocked primed template nucleic acid molecule. Morepreferably, the method further includes, after step (e), the steps of:(f) removing the reversible terminator moiety from the second blockedprimed template nucleic acid molecule to produce a second de-blockedprimer; and (g) incorporating a reversible terminator nucleotideincluding a reversible terminator moiety into the second de-blockedprimer to produce a third blocked primed template nucleic acid molecule.Still more preferably, steps (a)-(c) are repeated using the thirdblocked primed template nucleic acid molecule in place of the firstblocked primed template nucleic acid molecule. Optionally, in any of theembodiments step (c) occurs after all of the other steps have beenperformed. Optionally, in any of the embodiments none of the differenttest nucleotides includes an exogenous fluorescent label. Optionally, inany of the embodiments each of the different test nucleotides is adifferent native nucleotide. Optionally, in any of the embodiments thepolymerase does not include an exogenous fluorescent label, and step (b)does not include measuring any fluorescent signal from the polymerase.Optionally, in any of the embodiments the reaction mixture includes anon-catalytic metal ion that inhibits polymerase-mediated incorporation.Optionally, in any of the embodiments the reaction mixture includes acatalytic metal ion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are interferometry traces for the examination andincorporation cycles of the expected sequence (GAC) in examplesequencing runs. Binding signals for all four dNTPs at the same positionare illustrated in the presence of 0.1 mM MgCl₂ (FIG. 1A) or 1 mM MgCl₂(FIG. 1B). All examination cycles were conducted after incorporating thecorrect 3′-blocked nucleotide, but before cleavage of the 3′-ONH₂reversible terminator moiety to reveal an extendable 3′-OH group. Basesfor the next correct incoming nucleotides (n+1) are highlighted usingbold uppercase base identifiers for their respective positions in thesequence. Additionally, the second correct bases (n+2) are highlightedusing bold lowercase base identifiers on their respective binding signalpeaks. The order of nucleotide examination was: dATP, dTTP, dGTP, anddCTP.

FIG. 2 is an interferometry trace for the interrogation cycles of theexpected sequence (AGTG) showing all four dNTP binding signals for thesame position. All examination cycles were conducted after incorporatingthe correct aminooxy nucleotide, and then cleaving the 3′-ONH₂reversible terminator moiety to reveal an extendable 3′-OH group. Thenext correct incoming nucleotide (n+1) bases are highlighted in boldwith a larger font size for their respective positions in the sequence.Subsequent next correct bases are highlighted in bold with smaller fontsize on their respective binding signal peaks. The order of examinationwas dTTP, dATP, dGTP, and dCTP.

FIG. 3 is an interferometry trace for the interrogation cycles of theexpected sequence (TAGT) showing all four dNTP binding signals for thesame position. All interrogation cycles were conducted afterincorporating two aminooxy nucleotides with their respective cleavagereactions. All the next correct incoming nucleotide (n+1) bases arehighlighted in bold with larger font size for their respective positionsin the sequence. Moreover, the second correct bases are highlighted inbold with smaller font size on their respective binding signal peaks inthe graph. The interrogation order was dTTP, dATP, dGTP, and dCTP.

FIG. 4 is an interferometry trace for examination cycles of the expectedsequence of a GG homopolymer region. The polymerase showed only onebinding signal for dGTP since the sequence consisted of GG.

FIGS. 5A-5C are a series of graphs showing results from examination of aprimed template nucleic acid (having a free 3′-OH group) using pairwisecombinations of nucleotides. FIG. 5A is an interferometry trace showingmagnitude of binding (vertical axis) as a function of time (horizontalaxis), where different nucleotide pairs were used during examination.FIG. 5B is a bar graph showing the calculated R_(eq) parameter for eachdifferent nucleotide pair used in the examination step. FIG. 5C is a bargraph showing the calculated k_(obs), parameter for each differentnucleotide pair used in the examination step.

FIGS. 6A-6C are a series of graphs showing results from examination of aprimed template nucleic acid (having a 3′ reversibly terminatednucleotide) using pairwise combinations of nucleotides. FIG. 6A is aninterferometry trace showing magnitude of binding (vertical axis) as afunction of time (horizontal axis), where different nucleotide pairswere used during examination. FIG. 6B is a bar graph showing thecalculated R_(eq) parameter for each different nucleotide pair used inthe examination step. FIG. 6C is a bar graph showing the calculatedk_(obs) parameter for each different nucleotide pair used in theexamination step.

DETAILED DESCRIPTION

Provided herein is a method for identifying the next two cognatenucleotides downstream of a primer in a primed template nucleic acid,with or without a blocking moiety that precludes phosphodiester bondformation involving the 3′-OH group. The identification does not dependon incorporation of any nucleotide into the primer. The method can bepracticed using a sequencing-by-binding platform, and can involveexamination in the presence of any of non-catalytic metal ions thatinhibit polymerase-mediated incorporation, reversibly terminatedprimers, or the combination of reversibly terminated primers andcatalytic metal ions. Alternatively or additionally, examination can usea polymerase mutant that is catalytically inactive such that extensionis inhibited. Another means to prevent extension during an examinationstep is to use non-incorporable nucleotide analogs. Results from theidentification can be used for confirming sequencing information (e.g.,serving a verification function), or can facilitate incorporation of twonucleotides to extend the primer before any subsequent examination isperformed. When the primer is extended by incorporating two nucleotidespreliminary to the next examination step, the time needed to complete asequencing procedure can be reduced significantly.

In particular embodiments the method involves detecting the magnitudesof interaction between a primed template nucleic acid molecule(optionally having a blocked 3′-end), a polymerase, and a set of fourdifferent nucleotides without incorporating any of the nucleotides intothe primer of the primed template nucleic acid. More particularly, themethod can involve detecting signals indicating formation of a complexthat includes three molecular species and determining which two out ofthe possible four nucleotides are associated with the greatest amount ormagnitude of complex formation. The nucleotide giving the highest amountof complex formation (e.g., the highest magnitude of binding signal) isthe next correct nucleotide. The nucleotide giving the second-highestlevel of complex formation (e.g., the second-highest magnitude ofbinding signal) is the subsequent correct nucleotide.

Advantageously, the technique can even be practiced using various typesof nucleotides, including native (e.g., unlabeled) nucleotides,nucleotides with detectable labels (e.g., fluorescent or other opticallydetectable labels), or labeled or unlabeled nucleotide analogs (e.g.,modified nucleotides containing reversible terminator moieties).Further, the technique provides controlled reaction conditions,unambiguous determination of sequence, low overall cost of reagents, andlow instrument cost.

Indeed, numerous variations on the sequencing-by-binding assay chemistryare within the scope of alternatives that can be used for carrying outthe method described herein. Optionally, the primer of the primedtemplate nucleic acid molecule used in the examination step can be ablocked template nucleic acid molecule (e.g., as may result followingincorporation of a reversible terminator nucleotide into the primercomponent of the primed template nucleic acid molecule). Optionally,nucleotides used in the examination steps can include exogenous labels,such as exogenous fluorescent labels. Alternatively or additionally tothe use of labeled nucleotides, the polymerase used in the examinationsteps includes an exogenous fluorescent label. Optionally, neither thenucleotides nor the polymerase used in the examination steps includeexogenous detectable labels, and the system provides a route forlabel-free determination of the identities of two cognate nucleotidesfrom examination of four different nucleotides. Optionally, reversibleterminator nucleotides can be incorporated into a primer strand toensure that only a single nucleotide is incorporated. Optionally, a pairof reversible terminator nucleotides is incorporated into a primerstrand, with the reversible terminator moiety of the first reversibleterminator nucleotide being removed before the second reversibleterminator nucleotide is incorporated. Optionally, the reversibleterminator moiety is removed from the primer of a blocked primedtemplate nucleic acid before individuals from the next set of fourdifferent nucleotides are tested for binding in the presence of apolymerase. By this approach, the primer used in the examination stepswill terminate in a 3′-OH group that can participate in phosphodiesterbond formation with a cognate nucleotide. Optionally, the reversibleterminator moiety of the primer of a blocked primed template nucleicacid is left in place before individuals from the next set of fourdifferent nucleotides are tested for binding in the presence of apolymerase. By this approach, the blocked primer is used in theexamination reaction.

The technique may be applied to single nucleotide determination (e.g.,SNP determination based on a single full cycle procedure), oralternatively to more extensive nucleic acid sequencing proceduresemploying reiterative cycles that identify one nucleotide at a time. Forexample, the methods provided herein can be used in connection withsequencing-by-binding procedures, as described in the commonly ownedU.S. patent applications identified by Ser. Nos. 62/447,319 and2017/0022553, the disclosures of which are incorporated by referenceherein in their entireties.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. For clarity, the following specific terms have the specifiedmeanings. Other terms are defined in other sections herein.

The singular forms “a” “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Approximating language, as usedin the description and claims, may be applied to modify any quantitativerepresentation that could permissibly vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term such as “about” is not to be limited to the precisevalue specified. Unless otherwise indicated, all numbers expressingquantities of ingredients, properties such as molecular weight, reactionconditions, so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the compositions, apparatus, or methods of thepresent disclosure. At the very least, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

As used herein, “sequencing-by-binding” refers to a sequencing techniquewherein specific binding of a polymerase and a next correct nucleotideto a primed template nucleic acid is used for identifying the nextcorrect nucleotide to be incorporated into the primer strand of theprimed template nucleic acid. The specific binding interaction precedeschemical incorporation of the nucleotide into the primer strand, and soidentification of the next correct nucleotide can take place eitherwithout or before incorporation of the next correct nucleotide.

As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide”means at least two nucleotides covalently linked together. Thus, theterms include, but are not limited to, DNA, RNA, analogs (e.g.,derivatives) thereof or any combination thereof, that can be acted uponby a polymerizing enzyme during nucleic acid synthesis. The termincludes single-, double-, or multiple-stranded DNA, RNA and analogs(e.g., derivatives) thereof. Double-stranded nucleic acidsadvantageously can minimize secondary structures that may hinder nucleicacid synthesis. A double-stranded nucleic acid may possess a nick or asingle-stranded gap. A nucleic acid may represent a single, plural, orclonally amplified population of nucleic acid molecules.

As used herein, a “template nucleic acid” is a nucleic acid to bedetected, sequenced, evaluated or otherwise analyzed using a method orapparatus disclosed herein.

As used herein, a “primed template nucleic acid” (or alternatively,“primed template nucleic acid molecule”) is a template nucleic acidprimed with (i.e., hybridized to) a primer, wherein the primer is anoligonucleotide having a 3′-end with a sequence complementary to aportion of the template nucleic acid. The primer can optionally have afree 5′-end (e.g., the primer being noncovalently associated with thetemplate) or the primer can be continuous with the template (e.g., via ahairpin structure). The primed template nucleic acid includes thecomplementary primer and the template nucleic acid to which it is bound.A primed template nucleic acid molecule can be extendable in apolymerization reaction or it can be a blocked primed template nucleicacid. By “extendable” it is meant that a cognate nucleotide can bejoined to the 3′-end of a primer strand by formation of a phosphodiesterbond.

As used herein, a “blocked primed template nucleic acid” (oralternatively, “blocked primed template nucleic acid molecule”) is aprimed template nucleic acid modified to preclude or preventphosphodiester bond formation at the 3′-end of the primer. Blocking maybe accomplished, for example, by chemical modification with a blockinggroup at either the 3′ or 2′ position of the five-carbon sugar at the 3′terminus of the primer. Alternatively, or in addition, chemicalmodifications that preclude or prevent phosphodiester bond formation mayalso be made to the nitrogenous base of a nucleotide. Reversibleterminator nucleotide analogs including each of these types of blockinggroups will be familiar to those having an ordinary level of skill inthe art. Incorporation of these analogs at the 3′ terminus of a primerof a primed template nucleic acid molecule results in a blocked primedtemplate nucleic acid molecule. The blocked primed template nucleic acidincludes the complementary primer, blocked from extension at its 3′-end,and the template nucleic acid to which it is bound.

As used herein, a “nucleotide” is a molecule that includes a nitrogenousbase, a five-carbon sugar (e.g., ribose or deoxyribose), and at leastone phosphate group or functional analogs of such a molecule. Thefunctional analogs may have a function of forming a ternary complex witha polymerase and primed template nucleic acid (or blocked primedtemplate nucleic acid) and/or a function of being incorporated into aprimed template nucleic acid. The term embraces ribonucleotides,deoxyribonucleotides, nucleotides modified to include exogenous labelsor reversible terminators, and nucleotide analogs.

As used herein, a “native” nucleotide refers to a naturally occurringnucleotide that does not include an exogenous label (e.g., a fluorescentdye, or other label) or chemical modification such as may characterize anucleotide analog. Examples of native nucleotides useful for carryingout the sequencing-by-binding procedures described herein include: dATP(2′-deoxyadenosine-5′-triphosphate); dGTP(2′-deoxyguanosine-5′-triphosphate); dCTP(2′-deoxycytidine-5′-triphosphate); dTTP(2′-deoxythymidine-5′-triphosphate); and dUTP(2′-deoxyuridine-5′-triphosphate).

As used herein, a “nucleotide analog” has one or more modifications,such as chemical moieties, which replace, remove and/or modify any ofthe components (e.g., nitrogenous base, five-carbon sugar, or phosphategroup(s)) of a native nucleotide. Nucleotide analogs may be eitherincorporable or non-incorporable by a polymerase in a nucleic acidpolymerization reaction. Optionally, the 3′-OH group of a nucleotideanalog is modified with a moiety. The moiety may be a 3′ reversible orirreversible terminator of polymerase extension. The base of anucleotide may be any of adenine, cytosine, guanine, thymine, or uracil,or analogs thereof. Optionally, a nucleotide has an inosine, xanthine,hypoxanthine, isocytosine, isoguanine, nitropyrrole (including3-nitropyrrole) or nitroindole (including 5-nitroindole) base.Nucleotides may include, but are not limited to, ATP, UTP, CTP, GTP,ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dUTP, dCTP, dGTP,dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Nucleotides may alsocontain terminating inhibitors of DNA polymerase, dideoxynucleotides or2′,3′ dideoxynucleotides, which are abbreviated as ddNTPs (ddGTP, ddATP,ddTTP, ddUTP and ddCTP).

As used herein, the “next template nucleotide” (or the “next templatebase”) refers to the next nucleotide (or base) in a template nucleicacid that is located immediately downstream of the 3′-end of ahybridized primer.

As used herein, the term “next correct nucleotide” refers to thenucleotide type that will bind and/or incorporate at the 3′ end of aprimer to complement a base in a template strand to which the primer ishybridized. The base in the template strand is referred to as the “nexttemplate nucleotide” and is immediately 5′ of the base in the templatethat is hybridized to the 3′ end of the primer. The next correctnucleotide can be referred to as the “cognate” of the next templatenucleotide and vice versa. Cognate nucleotides that interact with eachother in a ternary complex or in a double stranded nucleic acid are saidto “pair” with each other. A nucleotide having a base that is notcomplementary to the next template base is referred to as an“incorrect”, “mismatch” or “non-cognate” nucleotide.

As used herein, a “blocking moiety,” when used with reference to anucleotide analog, is a part of the nucleotide that inhibits or preventsthe nucleotide from forming a covalent linkage to a second nucleotide(e.g., via the 3′-OH of a primer nucleotide) during the incorporationstep of a nucleic acid polymerization reaction. The blocking moiety of a“reversible terminator” nucleotide can be removed from the nucleotideanalog to allow for nucleotide incorporation. Such a blocking moiety isreferred to herein as a “reversible terminator moiety.” Exemplaryreversible terminator moieties are set forth in U.S. Pat. Nos.7,427,673; 7,414,116; 7,057,026; 7,544, 794 and 8,034,923; and PCTpublications WO 91/06678 and WO 07/123744, each of which is incorporatedby reference.

As used herein, a “test nucleotide” is a nucleotide being investigatedfor its ability to participate in formation of a ternary complex thatfurther includes a primed template nucleic acid and a polymerase.

As used herein, “polymerase” is a generic term for a protein or othermolecule that forms a ternary complex with a cognate nucleotide andprimed template nucleic acid (or blocked primed template nucleic acid)including but not limited to, DNA polymerase, RNA polymerase, reversetranscriptase, primase and transferase. Typically, the polymeraseincludes one or more active sites at which nucleotide binding may occur.Optionally a polymerase includes one or more active sites at whichcatalysis of nucleotide polymerization may occur. Optionally apolymerase lacks catalytic nucleotide polymerization function, forexample, due to a modification such as a mutation or chemicalmodification. Alternatively, the polymerase may catalyze thepolymerization of nucleotides to the 3′-end of a primer bound to itscomplementary nucleic acid strand. For example, a polymerase catalyzesthe addition of a next correct nucleotide to the 3′-OH group of theprimer via a phosphodiester bond, thereby chemically incorporating thenucleotide into the primer. Optionally, the polymerase used in theprovided methods is a processive polymerase. Optionally, the polymeraseused in the provided methods is a distributive polymerase.

As used herein, “biphasic” refers to a two-stage process wherein aprimed template nucleic acid is contacted with a polymerase and a testnucleotide. The first phase of the process involves contacting theprimed template nucleic acid with a polymerase in the presence of asub-saturating level of nucleotide(s), or even in the absence ofnucleotides. The term “sub-saturating” refers to a concentration belowthat required for achieving maximal binding at equilibrium. The secondphase of the process involves contacting the primed template nucleicacid from the first phase with a polymerase in the presence of a higherconcentration of nucleotide(s) than used in the first phase, where thehigher concentration is sufficient to yield maximal ternary complexformation when a nucleotide in the reaction is the next correctnucleotide.

As used herein, “providing” a template, a primer, a primed templatenucleic acid, or a blocked primed template nucleic acid refers to thepreparation or delivery of one or many nucleic acid polymers, forexample to a reaction mixture or reaction chamber.

As used herein, “monitoring” (or sometimes “measuring”), when used inreference to a molecular binding event, refers to a process of detectinga measurable interaction or binding between two molecular species. Forexample, monitoring may involve detecting measurable interactionsbetween a polymerase and primed template nucleic acid (or blocked primedtemplate nucleic acid), typically at various points throughout aprocedure. Monitoring can be intermittent (e.g., periodic) or continuous(e.g., without interruption), and can involve acquisition ofquantitative results. Monitoring can be carried out by detectingmultiple signals over a period of time during a binding event or,alternatively, by detecting signal(s) at a single time point during orafter a binding event.

As used herein, “contacting,” when used in reference to chemicalreagents, refers to the mixing together of reagents (e.g., mixing animmobilized template nucleic acid and either a buffered solution thatincludes a polymerase, or the combination of a polymerase and a testnucleotide) so that a physical binding reaction or a chemical reactionmay take place.

As used herein, “incorporating” or “chemically incorporating” or“incorporation,” when used in reference to a nucleic acid andnucleotide, refers to the process of joining a cognate nucleotide to anucleic acid primer by formation of a phosphodiester bond.

As used herein, a “binary complex” is a complex between a polymerase anda primed template nucleic acid (e.g., blocked primed template nucleicacid), where the complex does not include a nucleotide molecule such asthe next correct nucleotide.

As used herein, a “ternary complex” is a complex between a polymerase, aprimed template nucleic acid (e.g., blocked primed template nucleicacid), and the next correct nucleotide positioned immediately downstreamof the primer and complementary to the template strand of the primedtemplate nucleic acid or the blocked primed template nucleic acid. Theprimed template nucleic acid can include, for example, a primer with afree 3′-OH or a blocked primer (e.g., a primer with a chemicalmodification on the base or the sugar moiety of the 3′ terminalnucleotide, where the modification precludes enzymatic phosphodiesterbond formation).

As used herein, a “catalytic metal ion” refers to a metal ion thatfacilitates phosphodiester bond formation between the 3′-OH of a nucleicacid (e.g., a primer) and the phosphate of an incoming nucleotide by apolymerase. A “divalent catalytic metal cation” is a catalytic metal ionhaving a valence of two. Catalytic metal ions can be present atconcentrations necessary to stabilize formation of a complex between apolymerase, a nucleotide, and a primed template nucleic acid, referredto as non-catalytic concentrations of a metal ion. Catalyticconcentrations of a metal ion refer to the amount of a metal ionsufficient for polymerases to catalyze the reaction between the 3′-OHgroup of a nucleic acid (e.g., a primer) and the phosphate group of anincoming nucleotide.

As used herein, a “non-catalytic metal ion” refers to a metal ion that,when in the presence of a polymerase enzyme, does not facilitatephosphodiester bond formation needed for chemical incorporation of anucleotide into a primer. Typically, the non-catalytic metal ion is acation. A non-catalytic metal ion may inhibit phosphodiester bondformation by a polymerase, and so may stabilize a ternary complex bypreventing nucleotide incorporation. Non-catalytic metal ions mayinteract with polymerases, for example, via competitive binding comparedto catalytic metal ions. A “divalent non-catalytic metal ion” is anon-catalytic metal ion having a valence of two. Examples of divalentnon-catalytic metal ions include, but are not limited to, Ca²⁺, Zn²⁺,Co²⁺, Ni²⁺, and Sr²⁺. The trivalent Eu³⁺ and Tb³⁺ ions are non-catalyticmetal ions having a valence of three.

As used herein an “exogenous label” refers to a detectable chemicalmoiety of a sequencing reagent that is not present in a natural analogof the sequencing reagent, such as a non-naturally occurring labelpresent on a synthetic nucleotide analog or a synthetic polymeraseanalog (e.g., a DNA polymerase). While a native dNTP may have acharacteristic limited fluorescence profile, the native dNTP does notinclude any added colorimetric or fluorescent moiety. Conversely, a dATP(2′-deoxyadenosine-5′-triphosphate) molecule modified to include achemical linker and fluorescent moiety attached to the gamma phosphatewould be said to include an exogenous label because the attachedchemical components are not ordinarily a part of the nucleotide. Ofcourse, chemical modifications to add detectable labels to nucleotidebases also would be considered exogenous labels. Likewise, a DNApolymerase modified to include a fluorescent dye (e.g., by attachment toa cys residue that is part of the primary sequence of the enzyme) alsowould be said to include an exogenous label because the label is notordinarily a part of the polymerase.

As used herein, “unlabeled” refers to a molecular species free of addedor exogenous label(s) or tag(s). Of course, unlabeled nucleotides willnot include either of an exogenous fluorescent label, or an exogenousRaman scattering tag. A native nucleotide is another example of anunlabeled molecular species. An unlabeled molecular species can excludeone or more of the labels set forth herein or otherwise known in the artrelevant to nucleic acid sequencing or analytical biochemistry.

As used herein, “stabilize,” and its grammatical variants mean to holdsteady or limit disruption. “Stabilizing” a complex refers to promotingor prolonging the existence of the complex or inhibiting disruption ofthe complex. The term can be applied to any of a variety of complexesincluding, but not limited to a binary complex or ternary complex. Forexample, the complex that is stabilized can be a ternary complex betweena polymerase, primed template nucleic acid molecule (or blocked primedtemplate nucleic acid) and cognate nucleotide. Generally, stabilizationof the ternary complex prevents incorporation of the nucleotidecomponent of the ternary complex into the primed nucleic acid componentof the ternary complex. Accordingly, stabilizing a ternary complex canrefer to promoting or prolonging non-covalent interactions that bindcomponents of the ternary complex, or inhibiting disruption ofnon-covalent interactions that bind components of the ternary complex.

As used herein, the position of the 3′ terminal nucleotide of a primerrepresents position “N” or “n.” Thus, “N+1” refers to the position ofthe first cognate nucleotide to be incorporated into the primer, while“N+2” refers to the position of the second cognate nucleotide to beincorporated into the primer.

As used herein, “extension” refers to the process after anoligonucleotide primer and a template nucleic acid have annealed to oneanother, wherein a polymerase enzyme catalyzes addition of one or morenucleotides at the 3′-end of the primer. A nucleotide that is added to anucleic acid by extension is said to be “incorporated” into the nucleicacid. Accordingly, the term “incorporating” can be used to refer to theprocess of joining a nucleotide to the 3′-end of a primer by formationof a phosphodiester bond.

The terms “cycle” or “round,” when used in reference to a sequencingprocedure, refer to the portion of a sequencing run that is repeated toindicate the presence of a nucleotide. Typically, a cycle or roundincludes several steps such as steps for delivery of reagents, washingaway unreacted reagents and detection of signals indicative of changesoccurring in response to added reagents.

As used herein, a “ternary complex-stabilizing agent” is any agent thatpromotes or maintains stability of a ternary complex that includes:either a primed template nucleic acid molecule or a blocked primedtemplate nucleic acid molecule; a polymerase; and a cognate nucleotide(i.e., the next correct nucleotide). Examples include: a non-catalyticmetal ion that inhibits enzyme-mediated polymerization (e.g., Ca²⁺,Zn²⁺, Co²⁺, Ni²⁺, and Sr²⁺), including trivalent lanthanide cations(e.g., Eu³⁺ and Tb³⁺); polymerases engineered to have reduced capacityfor binary complex formation while exhibiting ternary complex formationcapacity; polymerases engineered for complete loss of ability tocatalyze phosphodiester bond formation in the presence of Mg²⁺ ion.

As used herein, “destabilize” and its grammatical variants mean to causesomething to be unable to continue existing or working in its usual way.“Destabilizing” a complex refers to the process of promoting dissolutionor breakdown of the complex (e.g., separation of the components of thecomplex). “Destabilizing” a complex also includes the process ofinhibiting or preventing formation of the complex. The term can beapplied to any of a variety of complexes including, but not limited to abinary complex or ternary complex. A ternary complex can be destabilizedin a way that does not necessarily result in formation of a covalentbond between a primed template nucleic acid and next correct nucleotide.For example, destabilization can result in dissociation of one or morecomponents from a ternary complex.

As used herein, the “magnitude of ternary complex formation” refers tothe measurable amount of a ternary complex that forms, where measurementmay involve qualitative assessment of binding curve height or shape; oralternatively quantitative assessment of curve height, time to reach amaximum binding (e.g., “saturation”), a value based on a mathematicalcurve fitting projection (e.g., related to a projected plateau level),and the like. A higher magnitude of ternary complex formation willindicate a greater number of ternary complexes formed.

Sequencing-by-Binding

Described herein are polymerase-based, nucleic acidsequencing-by-binding (SBB) reactions, wherein the polymerase undergoesconformational transitions between open and closed conformations duringdiscrete steps of the reaction. In one step, the polymerase binds to aprimed template nucleic acid to form a binary complex, also referred toherein as the pre-insertion conformation. In a subsequent step, anincoming nucleotide is bound and the polymerase fingers close, forming apre-chemistry conformation including a polymerase, primed templatenucleic acid and nucleotide; wherein the bound nucleotide has not beenincorporated. This step, also referred to herein as the “examination”step, may be followed by a chemical step wherein a phosphodiester bondis formed with concomitant pyrophosphate cleavage from the nucleotide(i.e., nucleotide incorporation). The polymerase, primed templatenucleic acid and newly incorporated nucleotide produce a post-chemistry,pre-translocation conformation. As both the pre-chemistry conformationand the pre-translocation conformation include a polymerase, primedtemplate nucleic acid and nucleotide, wherein the polymerase is in aclosed state, either conformation may be referred to herein as aclosed-complex or a closed ternary complex. In the closed pre-insertionstate, divalent catalytic metal ions, such as Mg²⁺ mediate a rapidchemical reaction involving nucleophilic displacement of a pyrophosphate(PPi) by the 3′ hydroxyl of the primer. The polymerase returns to anopen state upon the release of PPi, the post-translocation step, andtranslocation initiates the next round of reaction. While aclosed-complex can form in the absence of divalent catalytic metal ions(e.g., Mg²⁺), the polymerase of the closed-complex is proficient inchemical addition of nucleotide in the presence of the divalent metalions when provided with an appropriate substrate having an available3′-hydroxyl group. Low or deficient levels of catalytic metal ions, suchas Mg²⁺, lead to non-covalent (e.g., physical) sequestration of the nextcorrect nucleotide in a closed-complex. This closed-complex may bereferred to as a stabilized or trapped closed-complex. A stabilizedcomplex can also be formed using a catalytically inactive polymerasemutant or a polymerase that is complexed with a non-catalytic metal ion.Other means for forming a stabilized ternary complex include use of anon-incorporable nucleotide analog or use of a 3′ blocked primer in theternary complex. In any reaction step described above, the polymeraseconfiguration and/or interaction with a nucleic acid may be monitoredduring an examination step to identify the next correct base in thetemplate nucleic acid sequence. Before or after incorporation, reactionconditions can be changed to disengage the polymerase from the primedtemplate nucleic acid, and changed again to remove from the localenvironment any reagents that inhibit polymerase binding.

Generally speaking, a procedure disclosed herein can include a series offour or more “examination” steps that assess interaction between a testnucleotide and the next template base, and optionally an “incorporation”step that adds one or more complementary nucleotides (e.g., reversibleterminator nucleotides) to the 3′-end of the primer component of theprimed template nucleic acid molecule. Identity of the next correctnucleotide to be added is determined either without or before chemicallinkage of that nucleotide to the 3′-end of the primer through acovalent bond. The examination step can involve providing a primedtemplate nucleic acid to be used in the procedure, and contacting theprimed template nucleic acid with a polymerase enzyme (e.g., a DNApolymerase) and one or more test nucleotides being investigated as thepossible next correct nucleotide. Further, there is a step that involvesmonitoring or measuring the interaction between the polymerase and theprimed template nucleic acid in the presence of the test nucleotides.Optionally, the interaction can take place in the presence ofstabilizers, whereby the polymerase-nucleic acid interaction isstabilized in the presence of the next correct nucleotide. In someembodiments, the examination steps are capable of identifying ordetermining the identities of the two next correct nucleotides withoutrequiring incorporation of those nucleotides. Stated differently,identity of the next correct nucleotide, and the subsequent correctnucleotide, can be established without chemical incorporation of anynucleotide into the primer when one or more cycles of examination arecarried out using labeled or unlabeled nucleotides.

Whereas methods involving a single template nucleic acid molecule may bedescribed for convenience, these methods are merely illustrative. Thesequencing methods provided herein readily encompass a plurality oftemplate nucleic acids, wherein the plurality of nucleic acids may beclonally amplified copies of a single nucleic acid, or disparate nucleicacids, including combinations, such as populations of disparate nucleicacids that are clonally amplified. Thus, such sequencing methods arefully disclosed herein.

The Examination Step

An examination step according to the technique described hereintypically includes the following substeps: (1) providing a primedtemplate nucleic acid (i.e., a template nucleic acid molecule hybridizedwith a primer that optionally may be blocked from extension at its3′-end); (2) contacting the primed template nucleic acid with a reactionmixture that includes a polymerase and at least one test nucleotide; (3)monitoring the interaction of the polymerase with the primed templatenucleic acid molecule in the presence of the nucleotide and withoutchemical incorporation of any nucleotide into the primed templatenucleic acid; (4) repeating the process until binding reactions havebeen monitored for four different test nucleotides (e.g., dATP, dGTP,dCTP, and either dTTP or dUTP; or nucleotide analogs thereof); and (5)identifying the next two bases in the template nucleic acid (i.e., thenext correct nucleotide) using the monitored interactions. Optionally,the primed template nucleic acid molecule can be contacted initiallywith the polymerase in the absence of nucleotide before contacting anynucleotide. The primer of the primed template nucleic acid can be anextendible primer or a blocked primer. The primed template nucleic acid,the polymerase and the test nucleotide are capable of forming a ternarycomplex when the base of the test nucleotide is complementary to thenext base of the primed template nucleic acid molecule. The primedtemplate nucleic acid and the polymerase are capable of forming a binarycomplex when the base of the test nucleotide is not complementary to thenext base of the primed template nucleic acid molecule. Optionally, thecontacting occurs under conditions that favor formation of the ternarycomplex over formation of the binary complex. The identifying step caninclude identifying the base of the nucleotide that is complementary tothe next base of the primed template nucleic acid. Optionally, thisincludes contacting ternary complexes with one or more wash solutionshaving different nucleotide compositions that permit ternary complexesto be selectively maintained or dissociated.

All of these steps can be repeated one or more times to obtain extensivesequence information. For example, ternary complexes can be formedinitially by contacting a primed template nucleic acid (optionallyincluding a blocked 3′-end) with a polymerase (optionally labeled withan exogenous label) and, in serial fashion, four different nucleotides(optionally including one or more exogenous labels).

The examination step may be controlled so that nucleotide incorporationis either attenuated or accomplished. If nucleotide incorporation isattenuated during the examination step, then a separate incorporationstep may be performed after determining the identity of the next correctnucleotide. The separate incorporation step may be accomplished withoutthe need for monitoring, as the cognate nucleotide has already beenidentified during the examination step. If nucleotide incorporationproceeds during examination, subsequent nucleotide incorporation may beattenuated by use of a stabilizer that traps the polymerase on thenucleic acid after incorporation. A reversibly terminated nucleotide mayalso be used to prevent the addition of subsequent nucleotides. The SBBmethod allows for controlled determination of a template nucleic acidbase without requiring the use of labeled nucleotides, as theinteraction between the polymerase and template nucleic acid can bemonitored without a label on the nucleotide. To be clear, however, theuse of a labeled nucleotide (e.g., a fluorescent nucleotide) is optionalwhen performing the presently disclosed procedure to allow forfluorescent detection of bound nucleotide.

In the sequencing methods provided herein, the test nucleotideundergoing examination can include a 3′ hydroxyl group, or a blockingmoiety that prevents phosphodiester bond formation at the 3′-end of theprimer. A 3′ terminator moiety or a 2′ terminator moiety may be either areversible terminator or an irreversible terminator. Optionally, areversible terminator moiety is linked to the base of the 3′ nucleotideof the primer strand in the primed template nucleic acid molecule.Optionally, the reversible terminator of the at least one nucleotidemolecule is replaced or removed at some point after the examination stepthat employed the test nucleotide that included the reversibleterminator.

Contacting Steps

Contacting of the primed template nucleic acid molecule with reactionmixtures that include the polymerase and one or more test nucleotidemolecules can occur under conditions that stabilize formation of theternary complex and/or destabilize formation of the binary complex.Optionally, the reaction mixture includes potassium glutamate.Optionally, the conditions that stabilize formation of the ternarycomplex include contacting the primed template nucleic acid with astabilizing agent. Optionally, the reaction mixture includes astabilizing agent. The stabilizing agent can be one or morenon-catalytic metal ions that inhibit polymerase-mediated incorporation.Exemplary non-catalytic metal ions that inhibit polymerase-mediatedincorporation include strontium ion, tin ion, nickel ion, and europiumion. For example, the reaction mixture of the examination step thatincludes the primed template nucleic acid, the polymerase, and the testnucleotide also may include from 0.01 mM to 30 mM strontium chloride asa stabilizing agent.

Alternatively, and particularly when using a blocked primed templatenucleic acid to form a ternary complex in the examination step, reactionmixtures used for conducting examination and monitoring steps optionallycan include catalytic metal ions (e.g., Mg²⁺ or Mn²⁺). Concentrations ofthe catalytic metal ions needed to support polymerization activity whenusing unmodified (i.e., not 3′ blocked) primers will be familiar tothose having an ordinary level of skill in the art.

In certain embodiments, the primed template nucleic acid is immobilizedto the surface of a solid support. The immobilization may employ eithera covalent or a noncovalent bond between one or the other, or even bothstrands of the primed template nucleic acid and the solid support. Forexample, when the template and primer strands of the primed templatenucleic acid are different molecules, the template strand can beimmobilized, for example via its 5′-end. What is necessary, however, isthat the 3′ terminus of the primer is available for interacting with thepolymerase.

When the primed template nucleic acid is immobilized to a solid support,there are alternatives for how the contacting steps are performed. Forexample, the solid support can be physically transferred betweendifferent vessels (e.g., individual wells of a multiwell plate)containing different reagent solutions. This is convenientlyaccomplished using an automated or robotic instrument. In anotherexample, the primed template nucleic acid is immobilized to a solidsupport inside a flow cell or chamber. In this instance, differentcontacting steps can be executed by controlled flow of different liquidreagents through the chamber, or across the immobilized primed templatenucleic acid.

The Monitoring Step

Monitoring or measuring the interaction of the polymerase with theprimed template nucleic acid molecule in the presence of a nucleotidemolecule may be accomplished in many different ways. For example,monitoring can include measuring association kinetics for theinteraction between the primed template nucleic acid, the polymerase,and a nucleotide. Monitoring the interaction of the polymerase with theprimed template nucleic acid molecule in the presence of a nucleotidemolecule can include measuring equilibrium binding constants between thepolymerase and primed template nucleic acid molecule (i.e., equilibriumbinding constants of polymerase to the template nucleic acid in thepresence of a nucleotide). Thus, for example, the monitoring includesmeasuring the equilibrium binding constant of the polymerase to theprimed template nucleic acid in the presence of a nucleotide. Monitoringthe interaction of the polymerase with the primed template nucleic acidmolecule in the presence of a nucleotide molecule includes measuringdissociation kinetics of the polymerase from the primed template nucleicacid in the presence of any one of the four nucleotides. Optionally,monitoring the interaction of the polymerase with the primed templatenucleic acid molecule in the presence of a nucleotide molecule includesmeasuring kinetics of the dissociation of the closed complex (i.e.,dissociation of the primed template nucleic acid, the polymerase, andthe nucleotide). Optionally, the measured association kinetics differdepending on the identity of the nucleotide molecule. Optionally, thepolymerase has a different affinity for each type of nucleotideemployed. Optionally, the polymerase has a different dissociationconstant for each type of nucleotide in each type of closed-complex.Association, equilibrium and dissociation kinetics are known and can bereadily determined by one in the art. See, for example, Markiewicz etal., Nucleic Acids Research 40(16):7975-84 (2012); Xia et al., J. Am.Chem. Soc. 135(1):193-202 (2013); Brown et al., J. Nucleic Acids,Article ID 871939, 11 pages (2010); Washington, et al., Mol. Cell. Biol.24(2):936-43 (2004); Walsh and Beuning, J. Nucleic Acids, Article ID530963, 17 pages (2012); and Roettger, et al., Biochemistry47(37):9718-9727 (2008), which are incorporated by reference herein intheir entireties.

The monitoring step can include monitoring the steady state interactionof the polymerase with the primed template nucleic acid in the presenceof a first nucleotide, without chemical incorporation of the firstnucleotide into the primer of the primed template nucleic acid.Optionally, monitoring includes monitoring the dissociation of thepolymerase from the primed template nucleic acid in the presence of afirst nucleotide, without chemical incorporation of the first nucleotideinto the primer of the primed template nucleic acid. Optionally,monitoring includes monitoring the association of the polymerase withthe primed template nucleic acid in the presence of the firstnucleotide, without chemical incorporation of the first nucleotide intothe primer of the primed template nucleic acid. Again, test nucleotidesin these procedures may be native nucleotides (i.e., unlabeled), labelednucleotides (e.g., fluorescently labeled nucleotides), or nucleotideanalogs (e.g., nucleotides modified to include reversible orirreversible terminator moieties).

In the sequencing methods provided herein, the absence of a catalyticmetal ion in the reaction mixture or the absence of a catalytic metalion in the active site of the polymerase prevents the chemicalincorporation of the nucleotide into the primer of the primed templatenucleic acid. Optionally, the chelation of a catalytic metal ion in thereaction mixtures of the contacting step prevents the chemicalincorporation of the nucleotide into the primer of the primed templatenucleic acid. Optionally, a non-catalytic metal ion acts as a stabilizerfor the ternary closed-complex in the presence of the next correctnucleotide. Optionally, the substitution of a catalytic metal ion in thereaction mixtures of the contacting step with a non-catalytic metal ionprevents the chemical incorporation of the nucleotide molecule to theprimed template nucleic acid. Optionally, the catalytic metal ion ismagnesium. The metal ion mechanisms of polymerases postulates that a lowconcentration of metal ions may be needed to stabilize thepolymerase-nucleotide-DNA binding interaction. See, for instance,Section 27.2.2, Berg J M, Tymoczko J L, Stryer L, Biochemistry 5thEdition, WH Freeman Press, 2002.

Optionally, a low concentration of a catalytic ion in the reactionmixtures of the examination step (i.e., that are used for bindingpolymerase in the presence or absence of a test nucleotide) prevents thechemical incorporation of the test nucleotide into the primer of theprimed template nucleic acid. Optionally, a low concentration of thecatalytic ion (e.g., magnesium ion) is from about 1 μM to less than 100μM. Optionally, a low concentration is from about 0.5 μM to about 5 μM.Optionally, the reaction mixtures of the examination step includecobalt, and the incorporating step includes contacting with anincorporation reaction mixture containing a higher concentration ofcobalt as compared to the concentration of cobalt in the reactionmixtures of the examination step.

The examination step may be controlled, in part, by providing reactionconditions to prevent chemical incorporation of a nucleotide whileallowing monitoring of the interaction between the polymerase and theprimed template nucleic acid, thereby permitting determination of theidentity of the next base of the nucleic acid template strand. Suchreaction conditions may be referred to as “examination reactionconditions.” Optionally, a ternary complex or closed-complex is formedunder examination conditions. Optionally, a stabilized ternary complexor closed-complex is formed under examination conditions or in apre-chemistry conformation. Optionally, a stabilized closed-complex isin a pre-translocation conformation, wherein the enclosed nucleotide hasbeen incorporated, but the closed-complex does not allow for theincorporation of a subsequent nucleotide. Optionally, the examinationconditions accentuate the difference in affinity for polymerase toprimed template nucleic acids in the presence of different nucleotides.Optionally, the examination conditions cause differential affinity ofthe polymerase to the primed template nucleic acid in the presence ofdifferent nucleotides. By way of example, the examination conditionsthat cause differential affinity of the polymerase to the primedtemplate nucleic acid in the presence of different nucleotides include,but are not limited to, high salt and inclusion of potassium glutamate.Concentrations of potassium glutamate that can be used to alterpolymerase affinity for the primed template nucleic acid include 10 mMto 1.6 M of potassium glutamate, or any amount in between 10 mM and 1.6M. Optionally, high salt refers to a concentration of salt from 50 mM to1,500 mM salt.

Examination typically involves, in the monitoring step, detectingpolymerase interaction with a template nucleic acid, or with templatenucleic acid and nucleotide in combination. Detection may includeoptical, electrical, thermal, acoustic, chemical and mechanical means.Optionally, monitoring is performed after a buffer change or a washstep, wherein the wash step removes any non-bound reagents (e.g.,unbound polymerases and/or nucleotides) from the region of observation.Optionally, monitoring is performed during a buffer change or a washstep, such that the dissociation kinetics of the polymerase-nucleic acidor polymerase-nucleic acid-nucleotide complexes may be used to determinethe identity of the next base. Optionally, monitoring is performedduring the course of addition of the examination reaction mixture orfirst reaction mixture, such that the association kinetics of thepolymerase to the nucleic acid may be used to determine the identity ofthe next base on the nucleic acid. Optionally, monitoring involvesdistinguishing closed-complexes from binary complexes of polymerase andprimed template nucleic acid. Optionally, monitoring is performed underequilibrium conditions where the affinities measured are equilibriumaffinities. Multiple examination steps including different or similarexamination reagents, may be performed sequentially to ascertain theidentity of the next template base. Multiple examination steps may beutilized in cases where multiple template nucleic acids are beingsequenced simultaneously in one sequencing reaction, wherein differentnucleic acids react differently to the different examination reagents.Optionally, multiple examination steps may improve the accuracy of nextbase determination.

In an exemplary sequencing reaction, the examination step includesformation and/or stabilization of a closed-complex including apolymerase, a primed template nucleic acid, and the next correctnucleotide. Characteristics of the formation and/or release of theclosed-complex are monitored to identify the enclosed nucleotide andtherefore the next base in the template nucleic acid. Closed-complexcharacteristics can be dependent on the sequencing reaction components(e.g., polymerase, primer, template nucleic acid, nucleotide) and/orreaction mixture components and/or conditions. Optionally, theclosed-complex is in a pre-chemistry conformation. Optionally, theclosed-complex is in a pre-translocation conformation. Optionally, theclosed-complex is in a post-translocation conformation.

The examination step involves monitoring the interaction of a polymerasewith a primed template nucleic acid in the presence of a testnucleotide. The formation of a closed-complex may be monitored.Optionally, the absence of formation of a closed-complex is monitored.Optionally, the dissociation of a closed-complex is monitored.Optionally, the incorporation step involves monitoring incorporation ofa nucleotide. Optionally, the incorporation step involves monitoring theabsence of nucleotide incorporation.

Any process of the examination and/or incorporation step may bemonitored. Optionally, a polymerase has an exogenous label or “tag.”Optionally, the detectable tag or label on the polymerase is removable.Optionally, the nucleotides or polymerases have a detectable label,however, the label is not detected during sequencing. Optionally, nocomponent of the sequencing reaction is detectably labeled with anexogenous label.

Monitoring the variation in affinity of a polymerase for a templatenucleic acid in the presence of correct and incorrect nucleotides, underconditions that may or may not allow the incorporation of thenucleotide, may be used to determine the sequence of the nucleic acid.The affinity of a polymerase for a template nucleic acid in the presenceof different nucleotides, including modified or labeled nucleotides, canbe monitored as the off-rate of the polymerase-nucleic acid interactionin the presence of the various nucleotides. The affinities and off-ratesof many standard polymerases to various matched/correct,mismatched/incorrect and modified nucleotides are known in the art.Single molecule imaging of Klenow polymerase reveals that the off-ratefor a template nucleic acid for different nucleotide types, where thenucleotide types are prevented from incorporating, are distinctly andmeasurably different.

Optionally, a nucleotide of a particular type is made available to apolymerase in the presence of a primed template nucleic acid. Thereaction is monitored, wherein, if the nucleotide is a next correctnucleotide, the polymerase may be stabilized to form a closed-complex.If the nucleotide is an incorrect nucleotide, a closed-complex may stillbe formed; however, without the additional assistance of stabilizingagents or reaction conditions (e.g., absence of catalytic ions,polymerase inhibitors, salt), the closed-complex may dissociate. Therate of dissociation is dependent on the affinity of the particularcombination of polymerase, template nucleic acid, and nucleotide, aswell as reaction conditions. Optionally, the affinity is measured as anoff-rate. Optionally, the affinity is different between differentnucleotides for the closed-complex. For example, if the next base in thetemplate nucleic acid downstream of the 3′-end of the primer is G, thepolymerase-nucleic acid affinity, measured as an off-rate, is expectedto be different based on whether dATP, dCTP, dGTP or dTTP are added. Inthis case, dCTP would have the slowest off-rate, with the othernucleotides providing different off-rates for the interaction.Optionally, the off-rate may be different depending on the reactionconditions, for example, the presence of stabilizing agents (e.g.,absence of magnesium or inhibitory compounds) or reaction conditions(e.g., nucleotide modifications or modified polymerases). Once theidentity of the next correct nucleotide is determined, 1, 2, 3, 4 ormore nucleotide types may be introduced simultaneously to the reactionmixture under conditions that specifically target the formation of aclosed-complex. Excess nucleotides may be removed from the reactionmixture and the reaction conditions modulated to incorporate the nextcorrect nucleotide of the closed-complex. This sequencing reactionensures that only one nucleotide is incorporated per sequencing cycle.

The affinity of a polymerase for a template nucleic acid in the presenceof a nucleotide can be measured in a variety of methods known to one ofskill in the art. Optionally, the affinity is measured as an off-rate,where the off-rate is measured by monitoring the release of thepolymerase from the template nucleic acid as the reaction is washed by awash buffer. Optionally, the affinity is measured as an off-rate, wherethe off-rate is measured by monitoring the release of the polymerasefrom the template nucleic acid under equilibrium binding conditions,especially equilibrium binding conditions in which the polymerasebinding rates are low or diffusion limited. The polymerase binding ratesmay be diffusion limited at sufficiently low concentrations ofpolymerase, wherein if the polymerase falls off from the DNA-polymerasecomplex, it does not load back immediately, allowing for sufficient timeto detect that the polymerase has been released from the complex. For ahigher affinity interaction, the polymerase is released from the nucleicacid slowly, whereas a low affinity interaction results in thepolymerase being released more rapidly. The spectrum of affinities, inthis case, translates to different off-rates, with the off-ratesmeasured under dynamic wash conditions or at equilibrium. The smallestoff-rate corresponds to the base complementary to the added nucleotide,while the other off-rates vary, in a known fashion, depending on thecombination of polymerase and nucleotide selected.

Optionally, the off-rate is measured as an equilibrium signal intensityafter the polymerase and nucleotide are provided in the reactionmixture, wherein the interaction with the lowest off-rate (highestaffinity) nucleotide produces the strongest signal, while theinteractions with other, varying, off-rate nucleotides produce signalsof measurably different intensities. As a non-limiting example, afluorescently labeled polymerase, measured, preferably, under totalinternal reflection (TIRF) conditions, produces different measuredfluorescence intensities depending on the number of polymerase moleculesbound to surface-immobilized nucleic acid molecules in a suitably chosenwindow of time. The intrinsic fluorescence of the polymerase, forinstance, tryptophan fluorescence, may also be utilized. A high off-rateinteraction produces low measured intensities, as the number of boundpolymerase molecules, in the chosen time window is very small, wherein ahigh off-rate indicates that most of the polymerase is unbound from thenucleic acid. Any surface localized measurement scheme may be employedincluding, but not limited to, labeled or fluorescence schemes. Suitablemeasurement schemes that measure affinities under equilibrium conditionsinclude, but are not limited to, bound mass, refractive index, surfacecharge, dielectric constant, and other schemes known in the art.Optionally, a combination of on-rate and off-rate engineering yieldshigher fidelity detection in the proposed schemes. As a non-limitingexample, a uniformly low on-rate, base-dependent, varying off-rateresults in an unbound polymerase remaining unbound for prolongedperiods, allowing enhanced discrimination of the variation in off-rateand measured intensity. The on-rate may be manipulated by lowering theconcentration of the added polymerase, nucleotide, or both polymeraseand nucleotide.

Optionally, the interaction between the polymerase and the nucleic acidis monitored via a detectable tag attached to the polymerase. The tagmay be monitored by detection methods including, but limited to,optical, electrical, thermal, mass, size, charge, vibration, andpressure. The label may be magnetic, fluorescent or charged. Forexternal and internal label schemes, fluorescence anisotropy may be usedto determine the stable binding of a polymerase to a nucleic acid in aclosed-complex.

By way of example, a polymerase is tagged with a fluorophore, whereinternary complex formation is monitored as a stable fluorescent signal.The unstable interaction of the polymerase with the template nucleicacid in the presence of an incorrect nucleotide results in a measurablyweaker signal compared to the closed-complex formed in the presence ofthe next correct nucleotide. In certain preferred embodiments, however,the sequencing-by-binding procedure does not rely on detection of anyexogenous label (e.g., a fluorescent label) joined to the polymerase.For example, the polymerase can be a native polymerase.

Optionally, a primed template nucleic acid molecule (optionally blockedat its 3′-end) is contacted with polymerase and one exogenously labelednucleotide during each of the four or more examination steps used foreach cycle of determining identity of the next two cognate nucleotides.Monitoring of signal generated as a consequence of the presence of thelabeled nucleotide provides information concerning formation andstabilization/destabilization of the ternary complex that includes thelabeled nucleotide. For example, if the exogenous label is a fluorescentlabel, and if the primed template nucleic acid is immobilized to a solidsupport at a particular locus, then monitoring fluorescent signalassociated with that locus can be used for monitoring ternary complexformation and stability under different reaction mixture conditions. Insome embodiments, a primed template nucleic acid is contacted with apolymerase and a plurality of different exogenously labeled nucleotides.For example, at least 2, 3, 4 or more types of nucleotides can besimultaneously present. Optionally, different labels can be present fornucleotides having different base types, respectively.

The Identifying Step

The identity of the next correct base or nucleotide can be determined bymonitoring the presence, formation and/or dissociation of the ternarycomplex or closed-complex. The identities of the next two bases in thetemplate are determined without chemically incorporating the nextcorrect nucleotide into the 3′-end of the primer. Optionally, theidentities of the next two bases are determined by monitoring theaffinity of the polymerase for the primed template nucleic acid in thepresence of added nucleotides. Optionally, the affinity of thepolymerase for the primed template nucleic acid in the presence of thenext correct nucleotide may be used to determine the next correct baseon the template nucleic acid. Optionally, the affinity of the polymerasefor the primed template nucleic acid in the presence of an incorrectnucleotide may be used to determine the next correct base on thetemplate nucleic acid.

In certain embodiments, a ternary complex that includes a primedtemplate nucleic acid (or a blocked primed template nucleic acid) isformed in the presence of a polymerase and a plurality of nucleotides.Cognate nucleotide participating in the ternary complex optionally isidentified by observing destabilization of the complex that occurs whenthe cognate nucleotide is absent from the reaction mixture. This isconveniently carried out, for example, by exchanging one reactionmixture for another. Here, loss of the complex is an indicator ofcognate nucleotide identity. Loss of binding signal (e.g., a fluorescentbinding signal associated with a particular locus on a solid support)can occur when the primed template nucleic acid is exposed to a reactionmixture that does not include the cognate nucleotide. Optionally,maintenance of a ternary complex in the presence of a single nucleotidein a reaction mixture also can indicate identity of the cognatenucleotide.

The Incorporation Step

Optionally, the methods provided herein further include one or moreincorporation steps. By way of example, the incorporation step includesincorporating a single nucleotide (e.g., an unlabeled nucleotide, areversible terminator nucleotide, or a detectably labeled nucleotideanalog) complementary to the next base of the template nucleic acid intothe primer of the primed template nucleic acid molecule. Optionally, theincorporation step includes contacting the primed template nucleic acidmolecule, polymerase and nucleotide with an incorporation reactionmixture. The incorporation reaction mixture, typically includes acatalytic metal ion.

The provided method may further include preparing the primed templatenucleic acid molecule for a next examination step after theincorporation step. Optionally, the preparing includes subjecting theprimed template nucleic acid or the nucleic acid/polymerase complex toone or more wash steps; a temperature change; a mechanical vibration; apH change; salt or buffer composition changes, an optical stimulation ora combination thereof. Optionally, the wash step includes contacting theprimed template nucleic acid or the primed template nucleicacid/polymerase complex with one or more buffers, detergents, proteindenaturants, proteases, oxidizing agents, reducing agents, or otheragents capable of releasing internal crosslinks within a polymerase orcrosslinks between a polymerase and nucleic acid.

Optionally, the method further includes repeating the examination stepand the incorporation step to sequence a template nucleic acid molecule.The examination step may be repeated one or more times prior toperforming the incorporation step. Optionally, two consecutiveexamination steps include reaction mixtures with different nucleotidemolecules (e.g., different nucleotides that are labeled or unlabeled).Optionally, prior to incorporating the single nucleotide into the primedtemplate nucleic acid molecule, the first reaction mixture is replacedwith a second reaction mixture including a polymerase and 1, 2, 3, or 4types of nucleotide molecules (e.g., different unlabeled nucleotides).Optionally, the nucleotide molecules are native nucleotides selectedfrom dATP, dTTP, dCTP, and dGTP.

The incorporation reaction may be enabled by an incorporation reactionmixture. Optionally, the incorporation reaction mixture includes adifferent composition of nucleotides than the examination reaction. Forexample, the examination reaction includes one type of nucleotide andthe incorporation reaction includes another type of nucleotide. By wayof another example, the examination reaction includes one type ofnucleotide and the incorporation reaction includes four types ofnucleotides, or vice versa. Optionally, the examination reaction mixtureis altered or replaced by the incorporation reaction mixture.Optionally, the incorporation reaction mixture includes a catalyticmetal ion, potassium chloride, or a combination thereof.

Nucleotides present in the reaction mixture but not sequestered in aclosed-complex may cause multiple nucleotide insertions. Thus, a washstep can be employed prior to the chemical incorporation step to ensureonly the nucleotide sequestered within a trapped closed-complex isavailable for incorporation during the incorporation step. Optionally,free nucleotides may be removed by enzymes such as phosphatases. Thetrapped polymerase complex may be a closed-complex, a stabilizedclosed-complex or ternary complex involving the polymerase, primedtemplate nucleic acid and next correct nucleotide.

Optionally, the nucleotide enclosed within the closed-complex of theexamination step is incorporated into the 3′-end of the template nucleicacid primer during the incorporation step. Optionally, the nucleotideenclosed within the closed-complex of the examination step isincorporated during the examination step, but the closed-complex doesnot allow for the incorporation of a subsequent nucleotide; in thisinstance, the closed-complex is released during an incorporation step,allowing for a subsequent nucleotide to become incorporated.

Optionally, the incorporation step includes replacing a nucleotide fromthe examination step and incorporating another nucleotide into the3′-end of the template nucleic acid primer. The incorporation step canfurther involve releasing a nucleotide from within a closed-complex(e.g., the nucleotide is a modified nucleotide or nucleotide analog) andincorporating a nucleotide of a different kind to the 3′-end of thetemplate nucleic acid primer. Optionally, the released nucleotide isremoved and replaced with an incorporation reaction mixture including anext correct nucleotide.

Suitable reaction conditions for incorporation may involve replacing theexamination reaction mixture with an incorporation reaction mixture.Optionally, nucleotides present in the examination reaction mixture arereplaced with one or more nucleotides in the incorporation reactionmixture. Optionally, the polymerase present during the examination stepis replaced for the incorporation step. Optionally, the polymerasepresent during the examination step is modified for the incorporationstep. Optionally, the one or more nucleotides present during theexamination step are modified for the incorporation step. The reactionmixture and/or reaction conditions present during the examination stepmay be altered by any means for the incorporation step. These meansinclude, but are not limited to, removing reagents, chelating reagents,diluting reagents, adding reagents, altering reaction conditions such asconductivity or pH, and any combination thereof. The reagents in thereaction mixture including any combination of polymerase, primedtemplate nucleic acid, and nucleotide may be modified during theexamination step and/or incorporation step.

Optionally, the reaction mixture of the incorporation step includescompetitive inhibitors, wherein the competitive inhibitors reduce theoccurrence of multiple incorporations. In certain embodiments, thecompetitive inhibitor is a non-incorporable nucleotide. In certainembodiments, the competitive inhibitor is an aminoglycoside. Thecompetitive inhibitor is capable of replacing either the nucleotide orthe catalytic metal ion in the active site, such that after the firstincorporation the competitive inhibitor occupies the active sitepreventing a second incorporation. In some embodiments, both anincorporable nucleotide and a competitive inhibitor are introduced inthe incorporation step, such that the ratio of the incorporablenucleotide and the inhibitor can be adjusted to ensure incorporation ofa single nucleotide at the 3′-end of the primer.

Optionally, the provided reaction mixtures, including the incorporationreaction mixtures, include at least one unlabeled nucleotide moleculethat is a non-incorporable nucleotide. In other words, the providedreaction mixtures can include one or more unlabeled nucleotide moleculesthat are incapable of incorporation into the primer of the primedtemplate nucleic acid molecule. Nucleotides incapable of incorporationinclude, for example, diphosphate nucleotides. For instance, thenucleotide may contain modifications to the triphosphate group that makethe nucleotide non-incorporable. Examples of non-incorporablenucleotides may be found in U.S. Pat. No. 7,482,120, the disclosure ofwhich is incorporated by reference herein in its entirety. Optionally,the primer may not contain a free hydroxyl group at its 3′-end, therebyrendering the primer incapable of incorporating any nucleotide, and,thus making any nucleotide non-incorporable.

A polymerase inhibitor optionally may be included with the reactionmixtures containing test nucleotides in the examination step to trap thepolymerase on the nucleic acid upon binding the next correct nucleotide.Optionally, the polymerase inhibitor is a pyrophosphate analog.Optionally, the polymerase inhibitor is an allosteric inhibitor.Optionally, the polymerase inhibitor is a DNA or an RNA aptamer.Optionally, the polymerase inhibitor competes with a catalytic-ionbinding site in the polymerase. Optionally, the polymerase inhibitor isa reverse transcriptase inhibitor. The polymerase inhibitor may be anHIV-1 reverse transcriptase inhibitor or an HIV-2 reverse transcriptaseinhibitor. The HIV-1 reverse transcriptase inhibitor may be a(4/6-halogen/MeO/EtO-substituted benzo[d]thiazol-2-yl)thiazolidin-4-one.

In the provided sequencing methods, the next correct nucleotide isidentified before the incorporation step, allowing the incorporationstep to not require labeled reagents and/or monitoring. Thus, in theprovided methods, a nucleotide, optionally, does not contain an attacheddetectable tag or label. Optionally, the nucleotide contains adetectable label, but the label is not detected in the method.Optionally, the correct nucleotide does not contain a detectable label;however, an incorrect or non-complementary nucleotide to the next basecontains a detectable label. Optionally, the nucleotide contains adetectable label, that is detected during the examination step, howevera different nucleotide is incorporated during the incorporation step.

The examination step of the sequencing reaction may be repeated 1, 2, 3,4 or more times prior to the incorporation step. The examination andincorporation steps may be repeated until the desired sequence of thetemplate nucleic acid is obtained.

The formation of the closed-complex or the stabilized closed-complex canbe employed to ensure that only one nucleotide is added to the templatenucleic acid primer per cycle of sequencing, wherein the addednucleotide is sequestered within the closed-complex. The controlledincorporation of a single nucleotide per sequencing cycle enhancessequencing accuracy for nucleic acid regions including homopolymerrepeats.

Reaction Mixtures

Nucleic acid sequencing reaction mixtures, or simply “reactionmixtures,” typically include reagents that are commonly present inpolymerase-based nucleic acid synthesis reactions. Reaction mixturereagents include, but are not limited to, enzymes (e.g., thepolymerase), dNTPs, template nucleic acids, primer nucleic acids, salts,buffers, small molecules, co-factors, metals, and ions. The ions may becatalytic ions, divalent catalytic ions, non-catalytic ions that inhibitpolymerase-mediated incorporation, non-covalent metal ions, or acombination thereof. The reaction mixture can include salts such asNaCl, KCl, potassium acetate, ammonium acetate, potassium glutamate,NH₄Cl, or NH₄HSO₄. The reaction mixture can include a source of ions,such as Mg²⁺ or Mn²⁺ Mg-acetate, Co²⁺ or Ba²⁺. The reaction mixture caninclude tin ions, Ca²⁺, Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺, Ni²⁺, or Eu⁺³. Thebuffer can include Tris, Tricine, HEPES, MOPS, ACES, MES,phosphate-based buffers, and acetate-based buffers. The reaction mixturecan include chelating agents such as EDTA, EGTA, and the like.Optionally, the reaction mixture includes cross-linking reagents.Provided herein are reaction mixtures, optionally, used during theexamination step, as well as incorporation reaction mixtures used duringnucleotide incorporation that can include one or more of theaforementioned agents. Reaction mixtures, when used during examination,can be referred to herein as examination reaction mixtures. Optionally,the examination reaction mixture includes a high concentration of salt;a high pH; 1, 2, 3, 4, or more types of unlabeled nucleotides; potassiumglutamate; a chelating agent; a polymerase inhibitor; a catalytic metalion; a non-catalytic metal ion that inhibits polymerase-mediatedincorporation; or any combination thereof. The examination reactionmixture can include 10 mM to 1.6 M of potassium glutamate or any amountin between 10 mM and 1.6 M. Optionally, the incorporation reactionmixture includes a catalytic metal ion; 1, 2, 3, 4, or more types ofnucleotides (e.g., unlabeled nucleotides); potassium chloride; anon-catalytic metal ion; or any combination thereof.

Optionally, reaction mixtures in accordance with the disclosedtechniques modulate the formation and stabilization of a closed-complexduring an examination step. For example, the reaction conditions of theexamination step optionally can favor the formation and/or stabilizationof a closed-complex encapsulating a nucleotide, and hinder the formationand/or stabilization of a binary complex. The binary interaction betweenthe polymerase and template nucleic acid may be manipulated bymodulating sequencing reaction parameters such as ionic strength, pH,temperature, or any combination thereof, or by the addition of a binarycomplex destabilizing agent to the reaction. Optionally, high salt(e.g., 50 mM to 1,500 mM) and/or pH changes are utilized to destabilizea binary complex. Optionally, a binary complex may form between apolymerase and a template nucleic acid during the examination orincorporation step of the sequencing reaction, regardless of thepresence of a nucleotide. Optionally, the reaction conditions favor thestabilization of a closed ternary complex and destabilization of abinary complex. By way of example, the pH of the examination reactionmixture can be adjusted from pH 4.0 to pH 10.0 to favor thestabilization of a ternary complex and destabilization of a binarycomplex. Optionally, the pH of the examination reaction mixture is frompH 4.0 to pH 6.0. Optionally, the pH of the examination reaction mixtureis pH 6.0 to pH 10.0.

The provided reaction mixtures and sequencing methods disclosed hereinencourage polymerase interaction with the nucleotides and templatenucleic acid in a manner that reveals the identity of the next basewhile controlling the chemical addition of a nucleotide. Optionally, themethods are performed in the absence of detectably labeled nucleotidesor in the presence of labeled nucleotides wherein the labels are notdetected. Optionally, the reaction mixtures include nucleotides thatharbor an exogenous detectable label (e.g., a fluorescent label).Optionally, a plurality of nucleotides in a reaction mixture harbor thesame exogenous detectable label. Optionally, a plurality of nucleotidesin a reaction mixture harbor different exogenous detectable labels.Optionally, the reaction mixtures can include one or more exogenouslylabeled polymerase enzymes.

Provided herein are reaction mixtures and methods that facilitateformation and/or stabilization of a closed-complex that includes apolymerase bound to a primed template nucleic acid and a nucleotideenclosed within the polymerase-template nucleic acid complex, underexamination reaction mixture conditions. Examination reaction conditionsmay inhibit or attenuate nucleotide incorporation. Optionally,incorporation of the enclosed nucleotide is inhibited and the complex isstabilized or trapped in a pre-chemistry conformation or a ternarycomplex. Optionally, the enclosed nucleotide is incorporated andsubsequent nucleotide incorporation is inhibited. In this instance, thecomplex is stabilized or trapped in a pre-translocation conformation.For the sequencing reactions provided herein, the closed-complex isstabilized during the examination step, allowing for controllednucleotide incorporation. Optionally, a stabilized closed-complex is acomplex wherein incorporation of an enclosed nucleotide is attenuated,either transiently (e.g., to examine the complex and then incorporatethe nucleotide) or permanently (e.g., for examination only) during anexamination step. Optionally, a stabilized closed-complex allows for theincorporation of the enclosed nucleotide, but does not allow for theincorporation of a subsequent nucleotide. Optionally, the closed complexis stabilized in order to monitor any polymerase interaction with atemplate nucleic acid in the presence of a nucleotide for identificationof the next base in the template nucleic acid.

Optionally, the enclosed nucleotide has severely reduced or disabledbinding to the template nucleic acid in the closed-complex. Optionally,the enclosed nucleotide is base-paired to the template nucleic acid at anext base. Optionally, the identity of the polymerase, nucleotide,primer, template nucleic acid, or any combination thereof, affects theinteraction between the enclosed nucleotide and the template nucleicacid in the closed-complex.

Optionally, the enclosed nucleotide is bound to the polymerase of theclosed complex. Optionally, the enclosed nucleotide is weakly associatedwith the polymerase of the closed-complex. Optionally, the identity ofthe polymerase, nucleotide, primer, template nucleic acid, or anycombination thereof, affects the interaction between the enclosednucleotide and the polymerase in the closed-complex. For a givenpolymerase, each nucleotide has a different affinity for the polymerasethan another nucleotide. Optionally, this affinity is dependent, inpart, on the template nucleic acid and/or the primer.

The closed-complex may be transiently formed. Optionally, the enclosednucleotide is a next correct nucleotide. In some methods, the presenceof the next correct nucleotide contributes, in part, to thestabilization of a closed-complex. Optionally, the enclosed nucleotideis not a next correct nucleotide.

Optionally, the examination reaction condition comprises a plurality ofprimed template nucleic acids, polymerases, nucleotides, or anycombination thereof. Optionally, the plurality of nucleotides comprises1, 2, 3, 4, or more types of different nucleotides, for example dATP,dTTP, dGTP, and dCTP. Optionally, the plurality of template nucleicacids is a clonal population of template nucleic acids.

Reaction conditions that may modulate the stability of a closed-complexinclude, but are not limited to, the availability of catalytic metalions, suboptimal or inhibitory metal ions, ionic strength, pH,temperature, polymerase inhibitors, cross-linking reagents, and anycombination thereof. Reaction reagents which may modulate the stabilityof a closed-complex include, but are not limited to, non-incorporablenucleotides, incorrect nucleotides, nucleotide analogs, modifiedpolymerases, template nucleic acids with non-extendible polymerizationinitiation sites, and any combination thereof.

The examination reaction mixture can include other molecules including,but not limited to, enzymes. Optionally, the examination reactionmixture includes any reagents or biomolecules generally present in anucleic acid polymerization reaction. Reaction components may include,but are not limited to, salts, buffers, small molecules, metals, andions. Optionally, properties of the reaction mixture may be manipulated,for example, electrically, magnetically, and/or with vibration.

Nucleotides and Nucleotide Analogs

Nucleotides useful for carrying out the sequencing-by-binding proceduresdescribed herein include native nucleotides, labeled nucleotides (e.g.,nucleotides that include an exogenous fluorescent dye or other label notfound in native nucleotides), and nucleotide analogs (e.g., nucleotideshaving a reversible terminator moiety).

There is flexibility in the nature of the nucleotides that may beemployed in connection with the presently described technique. Anucleotide may include as its nitrogenous base any of: adenine,cytosine, guanine, thymine, or uracil. Optionally, a nucleotide includesinosine, xanthine, hypoxanthine, isocytosine, isoguanine, nitropyrrole(including 3-nitropyrrole) or nitroindole (including 5-nitroindole)base. Useful nucleotides include, but are not limited to, ATP, UTP, CTP,GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP,dUTP, dADP, dTDP, dCDP, dGDP, dUDP, dAMP, dTMP, dCMP, dGMP, and dUMP.Optionally, the phosphate group is modified with a moiety. The moietymay include a detectable label. Optionally, the 3′ OH group of thenucleotide is modified with a moiety, where the moiety may be a 3′reversible or irreversible terminator moiety. Optionally, the 2′position of the nucleotide is modified with a moiety, where the moietymay be a 2′ reversible or irreversible terminator moiety. Optionally,the base of the nucleotide is modified to include a reversibleterminator moiety. Nucleotides may also contain terminating inhibitorsof DNA polymerase, dideoxynucleotides or 2′,3′ dideoxynucleotides, whichare abbreviated as ddNTPs (ddGTP, ddATP, ddTTP, ddCTP, and ddUTP).

Optionally, a closed-complex of an examination step includes anucleotide analog or modified nucleotide to facilitate stabilization ofthe closed-complex. Optionally, a nucleotide analog includes anitrogenous base, five-carbon sugar, and phosphate group and anycomponent of the nucleotide may be modified and/or replaced. Nucleotideanalogs may be non-incorporable nucleotides. Non-incorporablenucleotides may be modified to become incorporable at any point duringthe sequencing method.

Nucleotide analogs include, but are not limited to, alpha-phosphatemodified nucleotides, alpha-beta nucleotide analogs, beta-phosphatemodified nucleotides, beta-gamma nucleotide analogs, gamma-phosphatemodified nucleotides, caged nucleotides, or ddNTPs. Examples ofnucleotide analogs are described in U.S. Pat. No. 8,071,755, which isincorporated by reference herein in its entirety.

Nucleotide analogs can include terminators that reversibly preventattachment of a subsequent nucleotide to the 3′-end of a primer that hasbeen extended with the nucleotide analog. One type of reversibleterminator is a 3′-O-blocked reversible terminator. Here the terminatormoiety is linked to the oxygen atom of the 3′-OH end of the 5-carbonsugar of a nucleotide. For example, U.S. Pat. Nos. 7,544,794 and8,034,923 (the disclosures of these patents are incorporated byreference) describe reversible terminator dNTPs having the 3′-OH groupreplaced by a 3′-ONH₂ group. Another type of reversible terminator is a3′-unblocked reversible terminator, wherein the terminator moiety islinked to the nitrogenous base of a nucleotide. For example, U.S. Pat.No. 8,808,989 (the disclosure of which is incorporated by reference)discloses particular examples of base-modified reversible terminatornucleotides that may be used in connection with the methods describedherein. Other reversible terminators that similarly can be used inconnection with the methods described herein include those described inU.S. Pat. Nos. 7,956,171, 8,071,755, and 9,399,798 (the disclosures ofthese U.S. patents are incorporated by reference). For reviews ofnucleotide analogs having terminators see e.g., Mu, R., et al., “TheHistory and Advances of Reversible Terminators Used in New Generationsof Sequencing Technology,” Genomics, Proteomics & Bioinformatics11(1):34-40 (2013). Optionally, one or more native nucleotides employedduring the examination step is replaced by a second type of nucleotidethat is incorporated during the incorporation step. For example,nucleotides present in the reaction mixture used during an examinationstep may be replaced by nucleotide analogs that include reversibleterminator moieties (e.g., positioned on the base or sugar of thenucleotide molecule).

Optionally, nucleotides are substituted for modified nucleotide analogshaving terminators that irreversibly prevent nucleotide incorporation tothe 3′-end of the primers that have incorporated the modified nucleotideanalogs. Irreversible nucleotide analogs include dideoxynucleotides,ddNTPs (ddGTP, ddATP, ddTTP, ddCTP). Dideoxynucleotides lack the 3′-OHgroup of dNTPs that is essential for polymerase-mediated synthesis.

Optionally, non-incorporable nucleotides include a blocking moiety thatinhibits or prevents the nucleotide from forming a covalent linkage to asecond nucleotide (3′ OH of a primer) during the incorporation step of anucleic acid polymerization reaction. The blocking moiety can be removedfrom the nucleotide after it has been incorporated into a primer byprimer extension, allowing for incorporation of a subsequent nucleotideinto the extended primer.

Optionally, a nucleotide analog present in a closed-complex renders theclosed-complex stable. Optionally, the nucleotide analog isnon-incorporable. Optionally, the nucleotide analog is released and anative nucleotide is incorporated. Optionally, the closed-complex isreleased, the nucleotide analog is modified, and the modified nucleotideanalog is incorporated. Optionally, the closed-complex is released underreaction conditions that modify and/or destabilize the nucleotide analogin the closed-complex.

Optionally, a nucleotide analog present in a closed-complex isincorporated and the closed-complex is stabilized. The closed-complexmay be stabilized by the nucleotide analog, or for example, by anystabilizing methods disclosed herein. Optionally, the nucleotide analogdoes not allow for the incorporation of a subsequent nucleotide. Theclosed-complex can be released, for example, by any methods describedherein, and the nucleotide analog is modified. The modified nucleotideanalog may allow for subsequent incorporation of a nucleotide to its3′-end.

Optionally, a nucleotide analog is present in the reaction mixtureduring the examination step. For example, 1, 2, 3, 4 or more nucleotideanalogs are present in the reaction mixture during the examination step.Optionally, a nucleotide analog is replaced, diluted, or sequesteredduring an incorporation step. Optionally, a nucleotide analog isreplaced with a native nucleotide. The native nucleotide may include anext correct nucleotide. Optionally, a nucleotide analog is modifiedduring an incorporation step. The modified nucleotide analog can besimilar to or the same as a native nucleotide.

Optionally, a nucleotide analog has a different binding affinity for apolymerase than a native nucleotide. Optionally, a nucleotide analog hasa different interaction with a next base than a native nucleotide.Nucleotide analogs and/or non-incorporable nucleotides may base-pairwith a complementary base of a template nucleic acid.

Optionally, a nucleotide analog is a nucleotide, modified or native,fused to a polymerase. Optionally, a plurality of nucleotide analogsincludes fusions to a plurality of polymerases, wherein each nucleotideanalog includes a different polymerase.

A nucleotide can be modified to favor the formation of a closed complexover the formation of a binary complex. A nucleotide may be selected ormodified to have a high affinity for a polymerase, wherein thepolymerase binds to a nucleotide prior to binding to the templatenucleic acid.

Any nucleotide modification that traps the polymerase in aclosed-complex may be used in the methods disclosed herein. Thenucleotide may be trapped permanently or transiently. Optionally, thenucleotide analog is not the means by which a closed complex isstabilized. Any closed-complex stabilization method may be combined in areaction utilizing a nucleotide analog.

Optionally, a nucleotide analog that allows for the stabilization of aclosed complex is combined with reaction conditions that usually releasethe closed-complex. The conditions include, but are not limited to, thepresence of a release reagent (e.g., catalytic metal ion, such asmagnesium or manganese). Optionally, the closed-complex is stabilizedeven in the presence of a catalytic metal ion. Optionally, theclosed-complex is released even in the presence of a nucleotide analog.Optionally, the stabilization of the closed-complex is dependent, inpart, on the concentrations and/or identity of the stabilization reagentand/or release reagents, and any combination thereof. Optionally, thestabilization of a closed-complex using nucleotide analogs is combinedwith additional reaction conditions that function to stabilize aclosed-complex, including, but not limited to, sequestering, removing,reducing, omitting, and/or chelating a catalytic metal ion; the presenceof a polymerase inhibitor, cross-linking agent; and any combinationthereof.

Optionally, one or more nucleotides can be labeled with distinguishingand/or detectable tags or labels; however, such tags or labels are notdetected during examination, identification of the base or incorporationof the base, and are not detected during the sequencing methodsdisclosed herein. The tags may be distinguishable by means of theirdifferences in fluorescence, Raman spectrum, charge, mass, refractiveindex, luminescence, length, or any other measurable property. The tagmay be attached to one or more different positions on the nucleotide, solong as the fidelity of binding to the polymerase-nucleic acid complexis sufficiently maintained to enable identification of the complementarybase on the template nucleic acid correctly. Optionally, the tag isattached to the nucleobase position of the nucleotide. Under suitablereaction conditions, the tagged nucleotides may be enclosed in aclosed-complex with the polymerase and the primed template nucleic acid.Alternatively, a tag is attached to the gamma phosphate position of thenucleotide.

Optionally, the labeled nucleotide can include 3-10 or more phosphategroups. Optionally, the labeled nucleotide can be any of adenosine,guanosine, cytidine, thymidine or uridine, or any other type of labelednucleotide. Optionally, the label can be an energy transfer acceptorreporter moiety. Optionally, the label can be a fluorescent dye.Optionally, the polymerase can be contacted with more than one type oflabeled nucleotide (e.g., A, G, C, and/or T/U, or others). Optionally,each type of labeled nucleotides can be operably linked to a differentreporter moiety to permit nucleotide identification. Optionally, eachtype of labeled nucleotide can be operably linked to the same type ofreporter moiety. Optionally, the labeled nucleotides are operably linkedat the terminal phosphate group with a reporter moiety. Optionally, thelabeled nucleotides are operably linked at the base moiety with areporter moiety. Optionally, the labeled nucleotide can be anon-incorporable nucleotide. Optionally, the non-incorporable nucleotidecan bind to the polymerase and primed template nucleic acid molecule ina template-dependent manner, but does not incorporate. Optionally,different types of labeled nucleotides can be employed in the method fordetecting the presence of a transiently-bound nucleotide in order todetermine the frequency, duration, or intensity, of a transiently-boundnucleotide. For example, a comparison can be made between thefrequency/duration/intensity of transiently-bound complementary andnon-complementary nucleotides. Under circumstances involving directexcitation of the reporter moiety, the length of the transient bindingtime of a complementary nucleotide can be longer and/or more frequentcompared to that of a non-complementary nucleotide.

Polymerases

Polymerases useful for carrying out the disclosed sequencing-by-bindingtechnique include naturally occurring polymerases and modified variantsthereof, including, but not limited to, mutants, recombinants, fusions,genetic modifications, chemical modifications, synthetics, and analogs.Naturally occurring polymerases and modified variants thereof are notlimited to polymerases that retain the ability to catalyze apolymerization reaction. Optionally, the naturally occurring and/ormodified variations thereof retain the ability to catalyze apolymerization reaction. Optionally, the naturally occurring and/ormodified variations have special properties that enhance their abilityto sequence DNA, including enhanced binding affinity to nucleic acids,reduced binding affinity to nucleic acids, enhanced catalysis rates,reduced catalysis rates etc. Mutant polymerases include polymeraseswherein one or more amino acids are replaced with other amino acids(naturally or non-naturally occurring), and insertions or deletions ofone or more amino acids.

Modified polymerases include polymerases that contain an external tag,which can be used to monitor the presence and interactions of thepolymerase. Optionally, intrinsic signals from the polymerase can beused to monitor their presence and interactions. Thus, the providedmethods can include monitoring the interaction of the polymerase,nucleotide and template nucleic acid through detection of an intrinsicsignal from the polymerase. Optionally, the intrinsic signal is a lightscattering signal. For example, intrinsic signals include nativefluorescence of certain amino acids such as tryptophan, wherein changesin intrinsic signals from the polymerase may indicate the formation of aternary-complex. Thus, in the provided methods, the polymerase can be anunlabeled polymerase and monitoring can be performed in the absence of adetectable label associated with the polymerase. Some modifiedpolymerases or naturally occurring polymerases, under specific reactionconditions, may incorporate only single nucleotides and may remain boundto the primer-template after the incorporation of the single nucleotide.Optionally, the thumb and finger domains of the polymerase may formtransient or covalent crosslinks due to their physical proximity in theclosed form of the polymerase. The crosslinks may be formed, for exampleby native or engineered cysteines at suitable positions on the thumb andfinger domains.

Optionally, the polymerase employed during the examination step includesone or more exogenous detectable label (e.g., a fluorescent label orRaman scattering tag) chemically linked to the structure of thepolymerase by a covalent bond. Optionally, the label(s) can be attachedafter the polymerase has been at least partially purified using proteinisolation techniques. For example, the exogenous detectable label can bechemically linked to the polymerase using a free sulfhydryl or a freeamine moiety of the polymerase. This can involve chemical linkage to thepolymerase through the side chain of a cysteine residue, or through thefree amino group of the N-terminus. An exogenous label can also beattached to a polymerase via protein fusion. Exemplary fluorescentlabels that can be attached via protein fusion include, for example,Green Fluorescent Protein (and wavelength shifted variants thereof) andphycobiliproteins (e.g., phycocyanin, phycoerythrin and variantsthereof). In certain preferred embodiments, a fluorescent label attachedto the polymerase is useful for locating the polymerase, as may beimportant for determining whether or not the polymerase has localized toa feature or spot on an array corresponding to immobilized primedtemplate nucleic acid. The fluorescent signal need not, and preferablydoes not change absorption or emission characteristics as the result ofbinding any nucleotide. Stated differently, the signal emitted by thelabeled polymerase can be maintained substantially uniformly in thepresence and absence of any nucleotide being investigated as a possiblenext correct nucleotide. In certain other preferred embodiments, thefluorescent signal emitted by the labeled polymerase is differentiallyaffected by inclusion of the polymerase in binary and ternary complexes.Labels useful in this regard are known to those having an ordinary levelof skill in the art.

The term polymerase and its variants, as used herein, also refers tofusion proteins including at least two portions linked to each other,for example, where one portion includes a peptide that can catalyze thepolymerization of nucleotides into a nucleic acid strand is linked toanother portion that includes a second moiety, such as, a reporterenzyme or a processivity-modifying domain. For example, T7 DNApolymerase includes a nucleic acid polymerizing domain and a thioredoxinbinding domain, wherein thioredoxin binding enhances the processivity ofthe polymerase. Absent the thioredoxin binding, T7 DNA polymerase is adistributive polymerase with processivity of only one to a few bases.Although DNA polymerases differ in detail, they have a similar overallshape of a hand with specific regions referred to as the fingers, thepalm, and the thumb; and a similar overall structural transition,including the movement of the thumb and/or finger domains, during thesynthesis of nucleic acids.

DNA polymerases include, but are not limited to, bacterial DNApolymerases, eukaryotic DNA polymerases, archaeal DNA polymerases, viralDNA polymerases and phage DNA polymerases. Bacterial DNA polymerasesinclude E. coli DNA polymerases I, II and III, IV and V, the Klenowfragment of E. coli DNA polymerase, Clostridium stercorarium (Cst) DNApolymerase, Clostridium thermocellum (Cth) DNA polymerase and Sulfolobussolfataricus (Sso) DNA polymerase. Eukaryotic DNA polymerases includeDNA polymerases α, β, γ, δ, €, η, ζ, λ, σ, μ, and k, as well as the Revlpolymerase (terminal deoxycytidyl transferase) and terminaldeoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNApolymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerases,PZA DNA polymerase, phi-15 DNA polymerase, Cp1 DNA polymerase, Cp7 DNApolymerase, T7 DNA polymerase, and T4 polymerase. Other DNA polymerasesinclude thermostable and/or thermophilic DNA polymerases such as DNApolymerases isolated from Thermus aquaticus (Taq) DNA polymerase,Thermus filiformis (Tfi) DNA polymerase, Thermococcus zilligi (Tzi) DNApolymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus flavusu(Tfl) DNA polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcusfuriosus (Pfu) DNA polymerase and Turbo Pfu DNA polymerase, Thermococcuslitoralis (Tli) DNA polymerase, Pyrococcus sp. GB-D polymerase,Thermotoga maritima (Tma) DNA polymerase, Bacillus stearothermophilus(Bst) DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, PfxDNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNA polymerase,Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus acidophiliumDNA polymerase; Sulfolobus acidocaldarius DNA polymerase; Thermococcussp. go N-7 DNA polymerase; Pyrodictium occultum DNA polymerase;Methanococcus voltae DNA polymerase; Methanococcus thermoautotrophicumDNA polymerase; Methanococcus jannaschii DNA polymerase; Desulfurococcusstrain TOK DNA polymerase (D. Tok Pol); Pyrococcus abyssi DNApolymerase; Pyrococcus horikoshii DNA polymerase; Pyrococcus islandicumDNA polymerase; Thermococcus fumicolans DNA polymerase; Aeropyrum pernixDNA polymerase; and the heterodimeric DNA polymerase DP1/DP2. Engineeredand modified polymerases also are useful in connection with thedisclosed techniques. For example, modified versions of the extremelythermophilic marine archaea Thermococcus species 9° N (e.g., TherminatorDNA polymerase from New England BioLabs Inc.; Ipswich, Mass.) can beused. Still other useful DNA polymerases, including the 3PDX polymeraseare disclosed in U.S. Pat. No. 8,703,461, the disclosure of which isincorporated by reference in its entirety.

RNA polymerases include, but are not limited to, viral RNA polymerasessuch as T7 RNA polymerase, T3 polymerase, SP6 polymerase, and Kllpolymerase; Eukaryotic RNA polymerases such as RNA polymerase I, RNApolymerase II, RNA polymerase III, RNA polymerase IV, and RNA polymeraseV; and Archaea RNA polymerases.

Reverse transcriptases include, but are not limited to, HIV-1 reversetranscriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2reverse transcriptase from human immunodeficiency virus type 2, MMLVreverse transcriptase from the Moloney murine leukemia virus, AMVreverse transcriptase from the avian myeloblastosis virus, andtelomerase reverse transcriptase that maintains the telomeres ofeukaryotic chromosomes.

Optionally, a polymerase is tagged with a chemiluminescent tag, whereinclosed complex formation is monitored as a stable luminescence signal inthe presence of the appropriate luminescence triggers. The unstableinteraction of the polymerase with the template nucleic acid in thepresence of an incorrect nucleotide results in a measurably weakersignal compared to the closed-complex formed in the presence of the nextcorrect nucleotide. Additionally, a wash step prior to triggeringluminescence could remove all polymerase molecules not bound in a stableclosed-complex.

Optionally, a polymerase is tagged with an optical scattering tag,wherein closed-complex formation is monitored as a stable opticalscattering signal. The unstable interaction of the polymerase with thenucleic acid in the presence of an incorrect nucleotide results in ameasurably weaker signal compared to the closed-complex formed in thepresence of the next correct nucleotide.

Optionally, the polymerase is tagged with a plasmonic nanoparticle tag,wherein the closed-complex formation is monitored as a shift inplasmonic resonance that is different from the plasmonic resonance inthe absence of the closed-complex or the presence of a closed-complexincluding an incorrect nucleotide. The change in plasmon resonance maybe due to the change in local dielectric environment in theclosed-complex, or it may be due to the synchronous aggregation of theplasmonic nanoparticles on a cluster of clonally amplified nucleic acidmolecules or another means that affects the plasmons differently in theclosed complex configuration.

Optionally, the polymerase is tagged with a Raman scattering tag,wherein the closed-complex formation is monitored as a stable Ramanscattering signal. The unstable interaction of polymerase with thenucleic acid in the presence of an incorrect nucleotide results in ameasurably weaker signal compared to the closed-complex formed in thepresence of the next correct nucleotide.

Optionally, the next two correct nucleotides are identified by a tag ona polymerase selected or modified to have a high affinity fornucleotides, wherein the polymerase binds to a nucleotide prior tobinding to the template nucleic acid. For example, the DNA polymerase Xfrom the African Swine Fever virus has an altered order of substratebinding, where the polymerase first binds to a nucleotide, then binds tothe template nucleic acid. Optionally, a polymerase is incubated witheach type of nucleotide in separate compartments, where each compartmentcontains a different type of nucleotide and where the polymerase islabeled differently with a tag depending on the nucleotide with which itis incubated. In these conditions, unlabeled nucleotides are bound todifferently labeled polymerases. The polymerases may be the same kind ofpolymerase bound to each nucleotide type or different polymerases boundto each nucleotide type. The differentially tagged polymerase-nucleotidecomplexes may be added simultaneously to any step of the sequencingreaction. Each polymerase-nucleotide complex binds to a template nucleicacid whose next base is complementary to the nucleotide in thepolymerase-nucleotide complex. The next two correct nucleotides areidentified by the tag on the polymerase carrying the nucleotides. Theinterrogation of the next template base by the labeledpolymerase-nucleotide complex may be performed under non-incorporatingand/or examination conditions, where once the identity of the nexttemplate base is determined, the complex is destabilized and removed,sequestered, and/or diluted and a separate incorporation step isperformed in a manner ensuring that only one nucleotide is incorporated.

A common method of introducing a detectable tag on a polymeraseoptionally involves chemical conjugation to amines or cysteines presentin the non-active regions of the polymerase. Such conjugation methodsare well known in the art. As non-limiting examples,n-hydroxysuccinimide esters (NHS esters) are commonly employed to labelamine groups that may be found on an enzyme. Cysteines readily reactwith thiols or maleimide groups, while carboxyl groups may be reactedwith amines by activating them with EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride).Optionally, N-hydroxysuccinimide (NHS) chemistry is employed at pHranges where only the N-terminal amines are reactive (for instance, pH7), such that only a single tag is added per polymerase.

Optionally, the tag attached to the polymerase is a charge tag, suchthat the formation of stable closed-complex can be detected byelectrical means by measuring changes in local charge density around thetemplate nucleic acids. Methods for detecting electrical charges arewell known in the art, including methods such as field-effecttransistors, dielectric spectroscopy, impedance measurements, and pHmeasurements, among others. Field-effect transistors include, but arenot limited to, ion-sensitive field-effect transistors (ISFET),charge-modulated field-effect transistors, insulated-gate field-effecttransistors, metal oxide semiconductor field-effect transistors andfield-effect transistors fabricated using semiconducting single wallcarbon nanotubes.

Optionally, a charge tag is a peptide tag having an isoelectric pointbelow about 4 or above about 10. Optionally, a polymerase including apeptide tag has a total isoelectric point below about 5 or above about9. A charge tag may be any moiety which is positively or negativelycharged. The charge tag may include additional moieties including massand/or labels such as dyes. Optionally, the charge tag possesses apositive or negative charge only under certain reaction conditions suchas changes in pH.

A polymerase may be labeled with a fluorophore and/or quencher.Optionally, a nucleic acid is labeled with a fluorophore and/orquencher. Optionally, one or more nucleotides are labeled with afluorophore and/or quencher. Exemplary fluorophores include, but are notlimited to, fluorescent nanocrystals; quantum dots; d-Rhodamine acceptordyes including dichloro[R110], dichloro[R6G], dichloro[TAMRA],dichloro[ROX] or the like; fluorescein donor dye including fluorescein,6-FAM, or the like; Cyanine dyes such as Cy3B; Alexa dyes, SETA dyes,Atto dyes such as atto 647N which forms a FRET pair with Cy3B and thelike. Fluorophores include, but are not limited to, MDCC(7-diethylamino-3-[([(2-maleimidyl)ethyl]amino)carbonyl]coumarin), TET,HEX, Cy3, TMR, ROX, Texas Red, Cy5, LC red 705 and LC red 640.Fluorophores and methods for their use including attachment topolymerases and other molecules are described in The Molecular Probes®Handbook (Life Technologies; Carlsbad Calif.) and Fluorophores Guide(Promega; Madison, Wis.), which are incorporated herein by reference intheir entireties. Exemplary quenches include, but are not limited to,ZEN, IBFQ, BHQ-1, BHQ-2, DDQ-I, DDQ-11, Dabcyl, Qxl quencher, Iowa BlackRQ, and IRDye QC-1.

Optionally, a conformationally sensitive dye may be attached close tothe active site of the polymerase without adversely affecting thepolymerization ability or fidelity of the polymerase; wherein a changein conformation, or a change in polar environment due to the formationof a closed-complex is reflected as a change in fluorescence orabsorbance properties of the dye. Common fluorophores such as Cy3 andfluorescein are known to have strong solvatochromatic response topolymerase binding and closed-complex formation, to the extent that theformation of closed-complex can be distinguished clearly from the binarypolymerase-nucleic acid complex. Optionally, the closed-complex can bedistinguished from binary complexes based on differences in fluorescenceor absorbance signals from a conformationally sensitive dye. Optionally,a solvatochromatic dye may be employed to monitor conformationaltransitions; wherein the change in local polar environment induced bythe conformational change can be used as the reporter signal.Solvatochromatic dyes include, but are not limited to, Reichart's dye,IR44, merocyanine dyes (e.g., merocyanine 540),4-[2-N-substituted-1,4-hydropyridin-4-ylidine)ethylidene]cyclohexa-2,5-dien-1-one,red pyrazolone dyes, azomethine dyes, indoaniline dyes, diazamerocyaninedyes, indigoid dyes, as exemplified by indigo, and others as well asmixtures thereof. Methods to introduce dyes or fluorophores to specificsites of a polymerase are well known in the art. As a non-limitingexample, a procedure for site specific labeling of a T7 DNA polymerasewith a dye is provided by Tsai et al., in “Site-Specific Labeling of T7DNA Polymerase with a Conformationally Sensitive Fluorophore and Its Usein Detecting Single-Nucleotide Polymorphisms,” Analytical Biochemistry384: 136-144 (2009), which is incorporated by reference herein in itsentirety.

Optionally, a polymerase is tagged with a fluorophore at a position thatcould sense closed-complex formation without interfering with thereaction. The polymerase may be a native or modified polymerase.Modified polymerases include those with one or more amino acidmutations, additions, and/or deletions. Optionally, one or more, but notall, cysteine amino acids are mutated to another amino acid, such asalanine. In this case, the remaining one or more cysteines are used forsite-specific conjugation to a fluorophore. Alternatively, one or moreamino acids are mutated to a reactive amino acid suitable forfluorophore conjugation, such as cysteines or amino acids includingprimary amines.

Optionally, binding between a polymerase and a template nucleic acid inthe presence of a correct nucleotide may induce a decrease influorescence, whereas binding with an incorrect nucleotide causes anincrease in fluorescence. Binding between a polymerase and a templatenucleic acid in the presence of a correct nucleotide may induce anincrease in fluorescence, whereas binding with an incorrect nucleotidecauses a decrease in fluorescence. The fluorescent signals may be usedto monitor the kinetics of a nucleotide-induced conformational changeand identify the next base in the template nucleic acid sequence.

Optionally, the polymerase/nucleic-acid interaction may be monitored byscattering signal originating from the polymerase or tags attached tothe polymerase, for instance, nanoparticle tags.

Conditions for Forming and Manipulating Closed-Complexes

As used herein, a closed-complex can be a ternary complex that includesa polymerase, primed template nucleic acid, and nucleotide. Theclosed-complex may be in a pre-chemistry conformation, wherein anucleotide is sequestered but not incorporated. The closed-complex mayalternatively be in a pre-translocation conformation, wherein anucleotide is incorporated by formation of a phosphodiester bond withthe 3′-end of the primer in the primed template nucleic acid. Theclosed-complex may be formed in the absence of catalytic metal ions ordeficient levels of catalytic metal ions, thereby physicallysequestering the next correct nucleotide within the polymerase activesite without chemical incorporation. Optionally, the sequesterednucleotide may be a non-incorporable nucleotide. The closed-complex maybe formed in the presence of catalytic metal ions, where theclosed-complex includes a nucleotide analog which is incorporated, but aPPi is not capable of release. In this instance, the closed-complex isstabilized in a pre-translocation conformation. Optionally, apre-translocation conformation is stabilized by chemically cross-linkingthe polymerase. Optionally, the closed-complex may be stabilized byexternal means. In some instances, the closed-complex may be stabilizedby allosteric binding of small molecules, or macromolecules such asantibodies or aptamers. Optionally, closed-complex may be stabilized bypyrophosphate analogs that bind close to the active site with highaffinity, preventing translocation of the polymerase.

As used herein, a stabilized closed-complex or stabilized ternarycomplex refers to a polymerase trapped at the polymerization initiationsite (3′-end of the primer) of the primed template nucleic acid by oneor a combinations of means, including but not limited to, crosslinkingthe thumb and finger domains in the closed conformation, binding of anallosteric inhibitor that prevents return of the polymerase to an openconformation, binding of pyrophosphate analogs that trap polymerase inthe pre-translocation step, absence of catalytic metal ions in theactive site of the polymerase, and addition of a metal ions such asnickel, tin and Sr²⁺ as substitutes for a catalytic metal ion. As such,the polymerase may be trapped at the polymerization initiation site evenafter the incorporation of a nucleotide. Therefore, the polymerase maybe trapped in the pre-chemistry conformation, pre-translocation step,post-translocation step or any intermediate step thereof. Thus, allowingfor sufficient examination and identification of the next correctnucleotide or base.

As described herein, a polymerase-based, sequencing-by-binding reactiongenerally involves providing a primed template nucleic acid with apolymerase and one or more of four different nucleotides in a serialfashion, wherein the nucleotides may or may not be complementary to thenext base of the primed template nucleic acid, and examining theinteraction of the polymerase with the primed template nucleic acidunder conditions wherein chemical incorporation of a nucleotide into theprimed template nucleic acid is disabled or severely inhibited in thepre-chemistry conformation. Optionally, wherein the pre-chemistryconformation is stabilized prior to nucleotide incorporation, preferablyusing stabilizers, a separate incorporation step may follow theexamination step to incorporate a single nucleotide to the 3′-end of theprimer. Optionally, where a single nucleotide incorporation occurs, thepre-translocation conformation may be stabilized to facilitateexamination and/or prevent subsequent nucleotide incorporation.

As indicated above, the presently described methods for sequencing anucleic acid include an examination step. The examination step involvesbinding a polymerase to the polymerization initiation site of a primedtemplate nucleic acid in a reaction mixture including at least one offour different nucleotides in a serial fashion, and monitoring theinteraction. Optionally, a nucleotide is sequestered within thepolymerase-primed template nucleic acid complex to form aclosed-complex, under conditions in which incorporation of the enclosednucleotide by the polymerase is attenuated or inhibited. Optionally astabilizer is added to stabilize the ternary complex in the presence ofthe next correct nucleotide. This closed-complex is in a stabilized orpolymerase-trapped pre-chemistry conformation. A closed-complex allowsfor the incorporation of the enclosed nucleotide but does not allow forthe incorporation of a subsequent nucleotide. This closed-complex is ina stabilized or trapped pre-translocation conformation. Optionally, thepolymerase is trapped at the polymerization site in its closed-complexby one or a combination of means including, but not limited to,crosslinking of the polymerase domains, crosslinking of the polymeraseto the nucleic acid, allosteric inhibition by small molecules,uncompetitive inhibitors, competitive inhibitors, non-competitiveinhibitors, and denaturation; wherein the formation of the trappedclosed-complex provides information about the identity of the next baseon the nucleic acid template.

Optionally, a closed-complex is released from its trapped or stabilizedconformation, which may allow for nucleotide incorporation to the 3′-endof the template nucleic acid primer. The closed-complex can bedestabilized and/or released by modulating the composition of thereaction conditions. In addition, the closed-complex can be destabilizedby electrical, magnetic, and/or mechanical means. Mechanical meansinclude mechanical agitation, for example, by using ultrasoundagitation. Mechanical vibration destabilizes the closed complex andsuppresses binding of the polymerase to the DNA. Thus, rather than awash step where the examination reaction mixture is replaced with anincorporation mixture, mechanical agitation may be used to remove thepolymerase from the template nucleic acid, enabling cycling throughsuccessive incorporation steps with a single nucleotide addition perstep.

Any combination of closed-complex stabilization or closed-complexrelease reaction conditions and/or methods may be combined. For example,a polymerase inhibitor that stabilizes a closed-complex may be presentin the examination reaction with a catalytic ion, which functions torelease the closed-complex. In the aforementioned example, the closedcomplex may be stabilized or released, depending on the polymeraseinhibitor properties and concentration, the concentration of thecatalytic metal ion, other reagents and/or conditions of the reactionmixture, and any combination thereof.

The closed-complex can be stabilized under reaction conditions wherecovalent attachment of a nucleotide to the 3′-end of the primer in theprimed template nucleic acid is attenuated. Optionally, theclosed-complex is in a pre-chemistry conformation or ternary complex.Optionally, the closed-complex is in a pre-translocation conformation.The formation of this closed-complex can be initiated and/or stabilizedby modulating the availability of a catalytic metal ion that permitsclosed-complex release and/or chemical incorporation of a nucleotide tothe primer in the reaction mixture. Exemplary metal ions include, butare not limited to, magnesium, manganese, cobalt, and barium. Catalyticions may be any formulation, for example, salts such as MgCl₂,Mg(CH₃CO₂)₂, and MnCl₂.

The selection and/or concentration of the catalytic metal ion may bebased on the polymerase and/or nucleotides in the sequencing reaction.For example, the HIV reverse transcriptase utilizes magnesium fornucleotide incorporation (N Kaushik, Biochemistry 35:11536-11546 (1996),and H P Patel, Biochemistry 34:5351-5363 (1995), which are incorporatedby reference herein in their entireties). The rate of closed-complexformation using magnesium versus manganese can be different depending onthe polymerase and the identity of the nucleotide. Thus, the stabilityof the closed complex may differ depending on catalytic metal ion,polymerase, and/or nucleotide identity. Further, the concentration ofcatalytic ion necessary for closed-complex stabilization may varydepending on the catalytic metal ion, polymerase, and/or nucleotideidentity and can be readily determined using the guidance providedherein. For example, nucleotide incorporation may occur at highcatalytic ion concentrations of one metal ion but does not occur at lowconcentrations of the same metal ion, or vice versa. Therefore,modifying metal ion identity, metal ion concentration, polymeraseidentity, and/or nucleotide identity allows for controlled examinationreaction conditions.

The closed-complex may be formed and/or stabilized by sequestering,removing, reducing, omitting, and/or chelating a catalytic metal ionduring the examination step of the sequencing reaction so thatclosed-complex release and/or chemical incorporation does not occur.Chelation includes any procedure that renders the catalytic metal ionunavailable for nucleotide incorporation, including using EDTA and/orEGTA. A reduction includes diluting the concentration of a catalyticmetal ion in the reaction mixture. The reaction mixture can be dilutedor replaced with a solution including a non-catalytic metal ion, whichpermits closed-complex formation, but inhibits nucleotide incorporation.Non-catalytic ions that inhibit polymerase-mediated incorporationinclude, but are not limited to, calcium, strontium, scandium, titanium,vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium,germanium, arsenic, selenium, rhodium, and strontium. Optionally, Ni²⁺is provided in an examination reaction to facilitate closed-complexformation. Optionally, Sr²⁺ is provided in an examination reaction tofacilitate closed-complex formation. Optionally, a non-catalytic metalion and a catalytic metal ion are both present in the reaction mixture,wherein one ion is present in a higher effective concentration than theother. In the provided methods, a non-catalytic ion such as cobalt canbecome catalytic (i.e., facilitate nucleotide incorporation) at highconcentrations. Thus, optionally, a low concentration of a non-catalyticmetal ion is used to facilitate ternary complex formation and a higherconcentration of the non-catalytic metal ion is used to facilitateincorporation.

Non-catalytic ions that inhibit polymerase-mediated incorporation may beadded to a reaction mixture under examination conditions. The reactionmay already include nucleotides. Optionally, non-catalytic ions arecomplexed to one or more nucleotides, and complexed nucleotides areadded to the reaction mixture. Non-catalytic ions can complex tonucleotides by mixing nucleotides with non-catalytic ions at elevatedtemperatures (about 80° C.). For example, a chromium nucleotide complexmay be added to a mixture to facilitate closed-complex formation andstabilization. Optionally, a chromium nucleotide complex is a chromiummonodentate, bidentate, or tridentate complex. Optionally, a chromiumnucleotide complex is an α-monodentate, or β-γ-bidentate nucleotide.

Optionally, a closed-complex is formed between a polymerase, primedtemplate nucleic acid, and nucleotide in reaction conditions includingSr²⁺, wherein Sr²⁺ promotes the formation of the closed-complex. Thepresence of Sr²⁺ can allow for the favorable formation of aclosed-complex including a next correct nucleotide over the formation acomplex including an incorrect nucleotide. The Sr²⁺ ion may be presentat concentrations from about 0.01 mM to about 30 mM. Optionally, Sr²⁺ ispresent as 10 mM SrCl₂. The formation of the closed-complex is monitoredunder examination conditions to identify the next base in the templatenucleic acid of the closed-complex. The affinity of the polymerase(e.g., Klenow fragment of E. coli DNA polymerase I, Bst) for each of thedNTPs (e.g., dATP, dTTP, dCTP, dGTP) in the presence of Sr²⁺ can bedifferent. Therefore, examination can involve measuring the bindingaffinities of polymerase-template nucleic acids to dNTPs; whereinbinding affinity is indicative of the next base in the template nucleicacid. Optionally, the binding interaction may be performed underconditions that destabilize the binary interactions between thepolymerase and primed template nucleic acid. Optionally, the bindinginteraction may be performed under conditions that stabilize the ternaryinteractions between the polymerase, the primed template nucleic acid,and the next correct nucleotide. After examination, a wash step removesunbound nucleotides, and Mg²⁺ is added to the reaction to inducepyrophosphate (PPi) cleavage and nucleotide incorporation. Optionally,the wash step includes Sr²⁺ to maintain the stability of the ternarycomplex, preventing the dissociation of the ternary complex. Thereaction may be repeated until a desired sequence read length isobtained.

Optionally, a closed-complex is formed between a polymerase, primedtemplate nucleic acid, and nucleotide in reaction conditions includingNi²⁺, wherein Ni²⁺ promotes the formation of the closed-complex. Thepresence of Ni²⁺ can allow for the favorable formation of aclosed-complex including a next correct nucleotide over the formation acomplex including an incorrect nucleotide. The Ni²⁺ ion may be presentat concentrations from about 0.01 mM to about 30 mM. Optionally, Ni²⁺ ispresent as 10 mM NiCl₂. The formation of the closed-complex is monitoredunder examination conditions to identify the next base in the templatenucleic acid of the closed-complex. The affinity of the polymerase(e.g., Klenow fragment of E. coli DNA polymerase I, Bst) for each of thedNTPs (e.g., dATP, dTTP, dCTP, dGTP) in the presence of Sr²⁺ can bedifferent. Therefore, examination can involve measuring the bindingaffinities of polymerase-template nucleic acids to dNTPs; whereinbinding affinity is indicative of the next base in the template nucleicacid. Optionally, the binding interaction may be performed underconditions that destabilize the binary interactions between thepolymerase and primed template nucleic acid. Optionally, the bindinginteraction may be performed under conditions that stabilize the ternaryinteractions between the polymerase, the primed template nucleic acid,and the next correct nucleotide. After examination, a wash removesunbound nucleotides and polymerase, and Mg²⁺ is added to the reaction toinduce pyrophosphate (PPi) cleavage and nucleotide incorporation.Optionally, the wash buffer includes Ni²⁺ to maintain the stability ofthe ternary complex, preventing the dissociation of the ternary complex.The reaction may be repeated until a desired sequence read length isobtained.

Optionally, a closed-complex is formed between a polymerase, primedtemplate nucleic acid, and nucleotide in reaction conditions includingnon-catalytic concentrations of Co²⁺, wherein Co²⁺ promotes theformation of the closed-complex. The presence of non-catalyticconcentrations of Co²⁺ can allow for the favorable formation of aclosed-complex including a next correct nucleotide over the formation acomplex including an incorrect nucleotide. The Co²⁺ ion may be presentat concentrations from about 0.01 mM to about 0.5 mM. Optionally, Co²⁺is present as 0.5 mM CoCl₂. The formation of the closed-complex ismonitored under examination conditions to identify the next base in thetemplate nucleic acid of the closed-complex. The affinity of thepolymerase (e.g., Klenow fragment of E. coli DNA polymerase I, Bst) foreach of the dNTPs (e.g., dATP, dTTP, dCTP, dGTP) in the presence of Co²⁺can be different. Therefore, examination can involve measuring thebinding affinities of polymerase-template nucleic acids to dNTPs;wherein binding affinity is indicative of the next base in the templatenucleic acid. Optionally, the binding interaction may be performed underconditions that destabilize the binary interactions between thepolymerase and primed template nucleic acid. Optionally, the bindinginteraction may be performed under conditions that stabilize the ternaryinteractions between the polymerase, the primed template nucleic acid,and the next correct nucleotide. After examination, a wash removesunbound nucleotides and polymerase, and Co²⁺ at a catalyticconcentration is added to the reaction to induce pyrophosphate (PPi)cleavage and nucleotide incorporation. Optionally, the wash bufferincludes non-catalytic amounts of Co²⁺ to maintain the stability of theternary complex, preventing the dissociation of the ternary complex. Thereaction may be repeated until a desired sequence read length isobtained.

Optionally, a catalytic metal ion may facilitate the formation of aclosed complex without subsequent nucleotide incorporation andclosed-complex release. Optionally, a concentration of 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 μM Mg²⁺ in a reaction mixture can induceconformational change of a polymerase to form a closed-complex withoutsubsequent nucleotide incorporation, PPi and closed-complex release.Optionally, the concentration of Mg²⁺ is from about 0.5 μM to about 10μM, from about 0.5 μM to about 5 μM, from about 0.5 μM to about 4 μM,from about 0.5 μM to about 3 μM, from about μM to about 5 μM, from about1 μM to about 4 μM, and from about 1 μM to about 3 μM.

Optionally, the concentration of available catalytic metal ion in thesequencing reaction which is necessary to allow nucleotide incorporationis from about 0.001 mM to about 10 mM, from about 0.01 mM to about 5 mM,from about 0.01 mM to about 3 mM, from about 0.01 mM to about 2 mM, fromabout 0.01 mM to about 1 mM, from about 0.05 mM to about 10 mM, fromabout 0.05 mM to about 5 mM, from about 0.05 mM to about 3 mM, fromabout 0.05 to about 2 mM, or from about 0.05 mM to about 1 mM.Optionally, the concentration of catalytic metal ion is from 5 mM to 50mM. Optionally, the concentration of catalytic metal ion is from 5 mM to15 mM, or about 10 mM.

A non-catalytic ion that inhibits polymerase-mediated incorporation maybe added to the reaction mixture at any stage including before, during,or after any of the following reaction steps: providing a primedtemplate nucleic acid, providing a polymerase, formation of a binarycomplex, providing a nucleotide, formation of a pre-chemistryclosed-complex, nucleotide incorporation, formation of apre-translocation closed-complex, and formation of a post-translocationconformation. The non-catalytic ion may be added to the reaction mixtureduring wash steps. The non-catalytic ion may be present through thereaction in the reaction mixture. For example, a catalytic ion is addedto the reaction mixture at concentrations which dilute the non-catalyticmetal ion, allowing for nucleotide incorporation.

The ability of catalytic and non-catalytic ions to modulate nucleotideincorporation may depend on conditions in the reaction mixtureincluding, but not limited to, pH, ionic strength, chelating agents,chemical cross-linking, modified polymerases, non-incorporablenucleotides, mechanical or vibration energy, and electric fields.

Optionally, the concentration of non-catalytic metal ion in thesequencing reaction necessary to allow for closed-complex formationwithout nucleotide incorporation is from about 0.1 mM to about 50 mM,from about 0.1 mM to about 40 mM, from about 0.1 mM to about 30 mM, fromabout 0.1 mM to about 20 mM, from about 0.1 mM to about 10 mM, fromabout 0.1 mM to about 5 mM, from about 0.1 to about 1 mM, from about 1mM to about 50 mM, from about 1 to about 40 mM, from about 1 mM to about30 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM,from about 1 mM to about 5 mM, from about 2 mM to about 30 mM, fromabout 2 mM to about 20 mM, from about 2 mM to about 10 mM, or anyconcentration within these ranges.

A closed-complex may be formed and/or stabilized by the addition of apolymerase inhibitor to the examination reaction mixture. Inhibitormolecules phosphonoacetate (phosphonoacetic acid) and phosphonoformate(phosphonoformic acid, common name Foscarnet), Suramin, Aminoglycosides,INDOPY-1 and Tagetitoxin are non-limiting examples of uncompetitive ornoncompetitive inhibitors of polymerase activity. The binding of theinhibitor molecule, near the active site of the enzyme, traps thepolymerase in either a pre-translocation or post-translocation step ofthe nucleotide incorporation cycle, stabilizing the polymerase in itsclosed-complex conformation before or after the incorporation of anucleotide, and forcing the polymerase to be bound to the templatenucleic acid until the inhibitor molecules are not available in thereaction mixture by removal, dilution or chelation.

Thus, provided is a method for sequencing a template nucleic acidmolecule that includes a series of examination steps, each includingproviding a template nucleic acid molecule primed with a primer;contacting the primed template nucleic acid molecule with a firstreaction mixture including a polymerase, a polymerase inhibitor and atleast one unlabeled nucleotide molecule; monitoring the interaction ofthe polymerase with the primed template nucleic acid molecule in thepresence of the unlabeled nucleotide molecule without incorporation ofthe nucleotide into the primer of the primed template nucleic acidmolecule; and identifying the nucleotide that is complementary to thenext base of the primed template nucleic acid molecule by the monitoredinteraction. The polymerase inhibitor prevents the incorporation of theunlabeled nucleotide molecule into the primer of the primer templatenucleic acid. Optionally, the inhibitor is a non-competitive inhibitor,an allosteric inhibitor, or an uncompetitive allosteric inhibitor.Optionally, the polymerase inhibitor competes with a catalytic ionbinding site in the polymerase.

Detection Platforms: Instrumentation for Detecting the Closed-Complex

The interaction between the polymerase and the template nucleic acid inthe presence of nucleotides can be monitored with or without the use ofan exogenous label. For example, the sequencing reaction may bemonitored by detecting the change in refractive index, fluorescenceemission, charge detection, Raman scattering detection, ellipsometrydetection, pH detection, size detection, mass detection, surface plasmonresonance, guided mode resonance, nanopore optical interferometry,whispering gallery mode resonance, nanoparticle scattering, photoniccrystal, quartz crystal microbalance, bio-layer interferometry,vibrational detection, pressure detection and other label-free detectionschemes that detect the added mass or refractive index due to polymerasebinding in a closed-complex with a template nucleic acid.

Optionally, detecting a change in refractive index is accomplished byone or a combination of means, including, but not limited to, surfaceplasmon resonance sensing, localized plasmon resonance sensing,plasmon-photon coupling sensing, transmission sensing throughsub-wavelength nanoholes (enhanced optical transmission), photoniccrystal sensing, interferometry sensing, refraction sensing, guided moderesonance sensing, ring resonator sensing, or ellipsometry sensing.Optionally, nucleic acid molecules may be localized to a surface,wherein the interaction of polymerase with nucleic acids in the presenceof various nucleotides may be measured as a change in the localrefractive index.

Optionally, the template nucleic acid is tethered to or localizedappropriately on or near a surface, such that the interaction ofpolymerase and template nucleic acid in the presence of nucleotideschanges the light transmitted across or reflected from the surface. Thesurface may contain nanostructures. Optionally, the surface is capableof sustaining plasmons or plasmon resonance. Optionally, the surface isa photonic substrate, not limited to a resonant cavity, resonant ring orphotonic crystal slab. Optionally, the surface is a guided moderesonance sensor. Optionally, the nucleic acid is tethered to, orlocalized appropriately on or near a nanohole array, a nanoparticle or amicroparticle, such that the interaction of polymerase and templatenucleic acid in the presence of nucleotides changes the absorbance,scattering, reflection or resonance of the light interacting with themicroparticle or nanoparticle.

Optionally, a nanohole array on a gold surface is used as a refractiveindex sensor. The template nucleic acid may be attached to a metalsurface by standard thiol chemistry, incorporating the thiol group onone of the primers used in a PCR reaction to amplify the DNA. When thedimensions of the nanohole array are appropriately tuned to the incidentlight, binding of the polymerase to the template nucleic acid in thepresence of nucleotides can be monitored as a change in lighttransmitted across the nanoholes. For both the labeled and label-freeschemes, simple and straightforward measurement of equilibrium signalintensity may reveal the formation of a stable closed complex.

Optionally, nucleic acid molecules are localized to a surface capable ofsustaining surface plasmons, wherein the change in refractive indexcaused by the polymerase interaction with localized nucleic acids may bemonitored through the change in the properties of the surface plasmons,wherein further, said properties of surface plasmons may include surfaceplasmon resonance. Surface plasmons, localized surface plasmons (LSP),or surface plasmon polaritons (SPP), arise from the coupling ofelectromagnetic waves to plasma oscillations of surface charges. LSPsare confined to nanoparticle surfaces, while SPPs and are confined tohigh electron density surfaces, at the interface between high electronmobility surfaces and dielectric media. Surface plasmons may propagatealong the direction of the interface, whereas they penetrate into thedielectric medium only in an evanescent fashion. Surface plasmonresonance conditions are established when the frequency of incidentelectromagnetic radiation matches the natural frequency of oscillationof the surface electrons. Changes in dielectric properties at theinterface, for instance due to binding or molecular crowding, affectsthe oscillation of surface electrons, thereby altering the surfaceplasmon resonance wavelength. Surfaces capable of surface plasmonresonance include, in a non-limiting manner, nanoparticles, clusters andaggregates of nanoparticles, continuous planar surfaces, nanostructuredsurfaces, and microstructured surfaces. Materials such as gold, silver,aluminum, high conductivity metal oxides (e.g., indium tin oxide, zincoxide, tungsten oxide) are capable of supporting surface plasmonresonance at their surfaces.

Optionally, a single nucleic acid molecule, or multiple clonal copies ofa nucleic acid, are attached to a nanoparticle, such that binding ofpolymerase to the nucleic acid causes a shift in the localized surfaceplasmon resonance (LSPR). Light incident on the nanoparticles inducesthe conduction electrons in them to oscillate collectively with aresonant frequency that depends on the nanoparticles' size, shape andcomposition. Nanoparticles of interest may assume different shapes,including spherical nanoparticles, nanorods, nanopyramids, nanodiamonds,and nanodiscs. As a result of these LSPR modes, the nanoparticles absorband scatter light so intensely that single nanoparticles are easilyobserved by eye using dark-field (optical scattering) microscopy. Forexample, a single 80-nm silver nanosphere scatters 445-nm blue lightwith a scattering cross-section of 3×10⁻² m², a million-fold greaterthan the fluorescence cross-section of a fluorescein molecule, and athousand fold greater than the cross-section of a similarly sizednanosphere filled with fluorescein to the self-quenching limit.Optionally, the nanoparticles are plasmon-resonant particles configuredas ultra-bright, nanosized optical scatters with a scattering peakanywhere in the visible spectrum. Plasmon-resonant particles areadvantageous as they do not bleach. Optionally, plasmon-resonantparticles are prepared, coated with template nucleic acids, and providedin a reaction mixture including a polymerase and one or morenucleotides, wherein a polymerase-template nucleic acid-particleinteraction is detected. One or more of the aforementioned steps may bebased on or derived from one or more methods disclosed by Schultz etal., in PNAS 97:996-1001 (2000), which is incorporated by referenceherein in its entirety.

The very large extinction coefficients at resonant wavelength enablesnoble-metal nanoparticles to serve as extremely intense labels fornear-surface interactions. Optionally, polymerase interaction withnanoparticle-localized DNA results in a shift in the resonantwavelength. The change in resonant wavelength due to binding or bindinginteractions can be measured in one of many ways. Optionally, theillumination is scanned through a range of wavelengths to identify thewavelength at which maximum scattering is observed at an imaging device.Optionally, broadband illumination is utilized in conjunction with adispersive element near the imaging device, such that the resonant peakis identified spectroscopically. Optionally, the nanoparticle system maybe illuminated at its resonant wavelength, or near its resonantwavelength, and any binding interactions may be observed as a drop inintensity of light scattered as the new resonant wavelength shifts awayfrom the illumination wavelength. Depending on the positioning of theilluminating wavelength, interactions may even appear as an increase innanoparticle scattering as the resonance peak shifts towards theillumination wavelength. Optionally, DNA-attached-nanoparticles may belocalized to a surface, or, alternatively, theDNA-attached-nanoparticles may be suspended in solution. A comprehensivereview of biosensing using nanoparticles is described by Anker et al.,in Nature Materials 7: 442-453 (2008), which is incorporated in itsentirety herein by reference.

Optionally, nano-features capable of LSPR are lithographically patternedon a planar substrate. The two dimensional patterning of nano-featureshas advantages in multiplexing and high-throughput analysis of a largenumber of different nucleic acid molecules. Optionally, gold nanopostsare substrates for surface plasmon resonance imaging detection ofpolymerase-template nucleic acid interactions, wherein the nucleic acidsare attached to the nanoposts. Nanostructure size and period caninfluence surface plasmon resonance signal enhancement, optionally,providing a 2, 3, 4, 5, 6, 7, 8-fold or higher signal amplification whencompared to control films.

Optionally, surface plasmon resonance may be sustained in planarsurfaces. A number of commercial instruments based on the Kretschmannconfiguration (e.g., Biacore, Uppsala, Sweden) and surface plasmonresonance imaging (e.g., GWC Technologies; Madison, Wis.; or Horiba;Kyoto, Japan) are available and have well established protocols forattaching DNA to their surfaces, as single spots and in multiplexedarray patterns. In the Kretschmann configuration, a metal film,typically gold, is evaporated onto the side of a prism and incidentradiation is launched at an angle to excite the surface plasmons. Anevanescent wave penetrates through the metal film exciting plasmons onthe other side, where it may be used to monitor near-surface and surfaceinteractions near the gold film. At the resonant angle, the lightreflected from the prism-gold interface is severely attenuated. Assumingfixed wavelength illumination, binding interactions may be examined bymonitoring both the intensity of the reflected light at a fixed angleclose to the resonant angle, as well as by monitoring the changes inangle of incidence required to establish surface plasmon resonanceconditions (minimum reflectivity). When a 2D imaging device such as aCCD or CMOS camera is utilized to monitor the reflected light, theentire illumination area may be imaged with high resolution. This methodis called surface plasmon resonance imaging (SPRi). It allows highthroughput analysis of independent regions on the surfacesimultaneously. Broadband illumination may also be used, in a fixedangle configuration, wherein the wavelength that is coupled to thesurface plasmon resonance is identified spectroscopically by looking fordips in the reflected spectrum. Surface interactions are monitoredthrough shifts in the resonant wavelength.

Surface plasmon resonance is a well-established method for monitoringprotein-nucleic acid interactions, and there exist many standardprotocols both for nucleic acid attachment as well as for analyzing thedata. Illustrative references from the literature include Cho et al.,“Binding Kinetics of DNA-Protein Interaction Using Surface PlasmonResonance,” Protocol Exchange, May 22, 2013; and Brockman et al., “AMultistep Chemical Modification Procedure To Create DNA Arrays on GoldSurfaces for the Study of Protein-DNA Interactions with Surface PlasmonResonance Imaging,” Journal of the American Chemical Society 121:8044-51 (1999), both of which are incorporated by reference herein intheir entireties.

Polymerase/nucleic-acid interactions may be monitored on nanostructuredsurfaces capable of sustaining localized surface plasmons. Optionally,polymerase/nucleic-acid interactions may be monitored on nanostructuredsurfaces capable of sustaining surface plasmon polaritons.

Optionally, polymerase/nucleic-acid interactions may be monitored onnanostructured surfaces capable of sustaining localized surfaceplasmons. Optionally, polymerase/nucleic-acid interactions may bemonitored on nanostructured surfaces capable of sustaining surfaceplasmon polaritons.

Optionally, extraordinary optical transmission (EOT) through a nanoholesarray may be used to monitor nucleic-acid/polymerase interactions. Lighttransmitted across subwavelength nanoholes in plasmonic metal films ishigher than expected from classical electromagnetic theory. Thisenhanced optical transmission may be explained by considering plasmonicresonant coupling to the incident radiation, whereby at resonantwavelength, a larger than anticipated fraction of light is transmittedacross the metallic nanoholes. The enhanced optical transmission isdependent on the dimensions and pitch of the nanoholes, properties ofthe metal, as well as the dielectric properties of the medium on eitherside of the metal film bearing the nanoholes. In the context of abiosensor, the transmissivity of the metallic nanohole array depends onthe refractive index of the medium contacting the metal film, whereby,for instance, the interaction of polymerase with nucleic acid attachedto the metal surface may be monitored as a change in intensity of lighttransmitted across the nanoholes array. Instrumentation and alignmentrequirements when using the EOT/plasmonic nanohole array approach ofsurface plasmon resonance may be employed using very compact optics andimaging elements. Low power LED illumination and a CMOS or CCD cameramay suffice to implement robust EOT plasmonic sensors. An exemplarynanohole array-based surface plasmon resonance sensing device isdescribed by Escobedo et al., in “Integrated Nanohole Array SurfacePlasmon Resonance Sensing Device Using a Dual-Wavelength Source,”Journal of Micromechanics and Microengineering 21: 115001 (2011), whichis herein incorporated by reference in its entirety.

The plasmonic nanohole array may be patterned on an optically opaquelayer of gold (greater than 50 nm thickness) deposited on a glasssurface. Optionally, the plasmonic nanohole array may be patterned on anoptically thick film of aluminum or silver deposited on glass.Optionally, the nanohole array is patterned on an optically thick metallayer deposited on low refractive index plastic. Patterning plasmonicnanohole arrays on low refractive index plastics enhances thesensitivity of the device to refractive index changes by better matchingthe refractive indices on the two sides of the metal layer. Optionally,refractive index sensitivity of the nanohole array is increased byincreasing the distance between holes. Optionally, nanohole arrays arefabricated by replication, for example, by embossing, casting,imprint-lithography, or template-stripping. Optionally, nanohole arraysare fabricated by self-assembly using colloids. Optionally, nanoholearrays are fabricated by projection direct patterning, such as laserinterference lithography.

A nano-bucket configuration may be preferable to a nanoholeconfiguration. In the nanohole configuration, the bottom of thenano-feature is glass or plastic or other appropriate dielectric,whereas in the nano-bucket configuration, the bottom of the nano-featureincludes a plasmonic metal. The nano-bucket array advantageously isrelatively simple to fabricate while maintaining the transmissionsensitivity to local refractive index.

Optionally, the nanohole array plasmonic sensing is combined withlens-free holographic imaging for large area imaging in an inexpensivemanner. Optionally, a plasmonic biosensing platform includes a plasmonicchip with nanohole arrays, a light-emitting diode source configured toilluminate the chip, and a CMOS imager chip to record diffractionpatterns of the nanoholes, which is modulated by molecular bindingevents on the surface. The binding events may be the formation of aclosed-complex between a polymerase and a template nucleic acid in thepresence of a nucleotide.

The methods to functionalize surfaces (for nucleic acid attachment) forsurface plasmon resonance sensing may be directly applied to EOTnanohole arrays as both sensing schemes employ similar metal surfaces towhich nucleic acids need to be attached.

Optionally, the refractive index changes associated withpolymerase/nucleic acid interaction may be monitored on nanostructuredsurfaces that do not support plasmons. Optionally, guided mode resonancemay be used to monitor the polymerase/nucleic-acid interaction.Guided-mode resonance or waveguide-mode resonance is a phenomenonwherein the guided modes of an optical waveguide can be excited andsimultaneously extracted by the introduction of a phase-matchingelement, such as a diffraction grating or prism. Such guided modes arealso called “leaky modes,” as they do not remain guided and have beenobserved in one and two-dimensional photonic crystal slabs. Guided moderesonance may be considered a coupling of a diffracted mode to awaveguide mode of two optical structured placed adjacent or on top ofeach other. For instance, for a diffraction grating placed on top of anoptical waveguide, one of the diffracted modes may couple exactly intothe guided mode of the optical waveguide, resulting in propagation ofthat mode along the waveguide. For off-resonance conditions, no light iscoupled into the waveguide, so the structure may appear completelytransparent (if dielectric waveguides are used). At resonance, theresonant wavelength is strongly coupled into the waveguide and may becouple out of the structure depending on downstream elements from thegrating-waveguide interface. In cases where the grating coupler isextended over the entire surface of the waveguide, the light cannot beguided, as any light coupled in is coupled out at the next gratingelement. Therefore, in a grating waveguide structure, resonance isobserved as a strong reflection peak, whereas the structure istransparent to off-resonance conditions. The resonance conditions aredependent on angle, grating properties, polarization and wavelength ofincident light. For cases where the guided mode propagation is notpresent, for instance due to a grating couple to the entire surface ofthe waveguide, the resonant mode may also be called leaky-moderesonance, in light of the strong optical confinement and evanescentpropagation of radiation in a transverse direction from the waveguidelayer. Change in dielectric properties near the grating, for instancedue to binding of biomolecules affects the coupling into the waveguide,thereby altering the resonant conditions. Optionally, where nucleic acidmolecules are attached to the surface of grating waveguide structures,the polymerase/nucleic-acid interaction may be monitored as a change inwavelength of the leaky mode resonance.

A diffraction element may be used directly on a transparent substratewithout an explicit need for a waveguide element. The change inresonance conditions due to interactions near the grating nanostructuremay be monitored as resonant wavelength shifts in the reflected ortransmitted radiation.

Reflected light from a nucleic acid attached guided mode resonant sensormay be used to monitor the polymerase/nucleic-acid interaction. Abroadband illumination source may be employed for illumination, and aspectroscopic examination of reflected light could reveal changes inlocal refractive index due to polymerase binding.

Optionally, a broadband illumination may be used and the transmittedlight may be examined to identify resonant shifts due to polymeraseinteraction. A linearly polarized narrow band illumination may be used,and the transmitted light may be filtered through a cross-polarizer;wherein the transmitted light is completely attenuated due to thecrossed polarizers excepting for the leaky mode response whosepolarization is modified. This implementation converts refractive indexmonitoring to a simple transmission assay that may be monitored oninexpensive imaging systems. Published material describe the assembly ofthe optical components. See, Nazirizadeh et al., “Low-Cost Label-FreeBiosensors Using Photonic Crystals Embedded between Crossed Polarizers,”Optics Express 18: 19120-19128 (2010), which is incorporated herein byreference in its entirety.

In addition to nanostructured surfaces, plain, unstructured surfaces mayalso be used advantageously for monitoring refractive index modulations.Optionally, interferometry may be employed to monitor the interaction ofpolymerase with nucleic acid bound to an un-structured, opticallytransparent substrate. Nucleic acid molecules may be attached to the topsurface of a glass slide by any means known in the art, and the systemilluminated from the bottom surface of the glass slide. There are tworeflection surfaces in this configuration, one reflection from thebottom surface of the glass slide, and the other from the top surfacewhich has nucleic acid molecules attached to it. The two reflected wavesmay interfere with each other causing constructive or destructiveinterference based on the path length differences, with the wavereflected from the top surface modulated by the changes in dielectricconstant due to the bound nucleic acid molecules (and subsequently bythe interaction of polymerase with the bound nucleic acid molecules).With the reflection from the bottom surface unchanged, any binding tothe nucleic acid molecules may be reflected in the phase differencebetween the beams reflected from the top and bottom surfaces, which inturn affects the interference pattern that is observed. Optionally,bio-layer interferometry is used to monitor the nucleic acid/polymeraseinteraction. Bio-layer interferometry may be performed on commercialdevices such as those sold by Pall Forte Bio corporation (Menlo Park,Calif.).

Optionally, the reflected light from the top surface is selectivelychosen by using focusing optics. The reflected light from the bottomsurface is disregarded because it is not in the focal plane. Focusingonly on the nucleic-acid-attached top surface, the light collected bythe focusing lens includes a planar wave, corresponding to the partiallyreflected incident radiation, and a scattered wave, corresponding to theradiations scattered in the collection direction by molecules in thefocal plane. These two components may be made to interfere if theincident radiation is coherent. This scattering based interferometricdetection is extremely sensitive and can be used to detect down tosingle protein molecules.

Optionally, a field-effect transistor (FET) is configured as a biosensorfor the detection of a closed-complex. A gate terminal of the FET ismodified by the addition of template nucleic acids. The binding of apolymerase including a charged tag results in changes in electrochemicalsignals. Binding of a polymerase with a next correct nucleotide to thetemplate nucleic acid provides different signals than polymerase bindingto a template nucleic acid in the presence of other incorrectnucleotides, where each incorrect nucleotide may also provide adifferent signal. Optionally, polymerase interactions with a templatenucleic acid are monitored using FET without the use of an exogenouslabel on the polymerase, primed template nucleic acid, or nucleotide.Optionally, the pH change that occurs due to release of H⁺ ions duringthe incorporation reaction is detected using a FET. Optionally, thepolymerase includes a tag that generates continuous H⁺ ions that isdetected by the FET. Optionally, the continuous H⁺ ion generating tag isan ATP synthase. Optionally, the continuous H⁺ ion generation tag ispalladium, copper or another catalyst. Optionally, the release of a PPiafter nucleotide incorporation is detected using FET. For example, onetype of nucleotide may be provided to a reaction at a time. Once thenext correct nucleotide is added and conditions allow for incorporation,PPi is cleaved, released, and detected using FET, therefore identifyingthe next correct nucleotide and the next base. Optionally, templatenucleic acids are bound to walls of a nanotube. Optionally, a polymeraseis bound to a wall of a nanotube. FET is advantageous for use as asequencing sensor due to its small size and low weight, making itappropriate for use as a portable sequencing monitoring component.Details of FET detection of molecular interactions are described by Kimet al., in “An FET-Type Charge Sensor for Highly Sensitive Detection ofDNA Sequence,” Biosensors and Bioelectronics, Microsensors andMicrosystems 20: 69-74 (2004), doi:10.1016/j.bios.2004.01.025; and byStar et al., in “Electronic Detection of Specific Protein Binding UsingNanotube FET Devices,” Nano Letters 3: 459-63 (2003),doi:10.1021/n10340172, which are incorporated by reference herein intheir entireties.

By way of example, the polymerase includes a fluorescent tag. To monitorpolymerase-nucleic acid interaction with high signal-to-noise,evanescent illumination or confocal imaging may be employed. Theformation of a closed-complex on localized template nucleic acids may beobserved as an increased fluorescence compared to the background, forinstance, whereas in some instances it may also be observed as adecreased fluorescence due to quenching or change in local polarenvironment. Optionally, a fraction of polymerase molecules may betagged with a fluorophore while another fraction may be tagged with aquencher in the same reaction mixture; wherein, the formation ofclosed-complex on a localized, clonal population of nucleic acid isrevealed as decrease in fluorescence compared to the background. Theclonal population of nucleic acids may be attached to a support surfacesuch as a planar substrate, microparticle, or nanoparticle. Optionally,a polymerase is tagged with a fluorophore, luminophore,chemiluminophore, chromophore, or bioluminophore.

Optionally, a plurality of template nucleic acids is tethered to asurface and one (or more) dNTPs are flowed in sequentially. The spectrumof affinities reveals the identity of the next correct nucleotide andtherefore the next base in the template nucleic acid. Optionally, theaffinities are measured without needing to remove and replace reactionmixture conditions (i.e., a wash step). Autocorrelation of the measuredintensities of the binding interaction, for instance, could readilyreveal the dynamics of nucleic acid sequence. Optionally, examinationincludes monitoring the affinity of the polymerase to the primedtemplate nucleic acid in the presence of nucleotides. Optionally, thepolymerase binds transiently with the nucleic acid and the bindingkinetics and affinity provides information about the identity of thenext base on the template nucleic acid. Optionally, a closed complex isformed, wherein the reaction conditions involved in the formation of theclosed complex provide information about the next base on the nucleicacid.

Any technique that can measure dynamic interactions between a polymeraseand nucleic acid may be used to measure the affinities and enable thesequencing reaction methods disclosed herein.

Systems for Detecting Nucleotide-Specific Ternary Complex Formation

The provided methods can be performed using a platform, where anycomponent of the nucleic acid polymerization reaction is localized to asurface. Optionally, the template nucleic acid is attached to a planarsubstrate, a nanohole array, a microparticle, or a nanoparticle.Optionally, all reaction components are freely suspended in the reactionmixture, and not immobilized to a solid support substrate.

Optionally, the template nucleic acid is immobilized to a surface. Thesurface may be a planar substrate, a hydrogel, a nanohole array, amicroparticle, or a nanoparticle. Optionally, the reaction mixturescontain a plurality of clonally amplified template nucleic acidmolecules. Optionally, the reaction mixtures contain a plurality ofdistinguishable template nucleic acids.

Provided herein, inter alia, are systems for performing sequencingreactions involving the examination of the interaction between apolymerase and a primed template nucleic acid in the presence ofnucleotides to identify the next base in the template closed-complex byone or a combination of means including, but not limited to,crosslinking of the polymerase domains, crosslinking of the polymeraseto the nucleic acid, allosteric inhibition by small molecules,uncompetitive inhibitors, competitive inhibitors, non-competitiveinhibitors, and denaturation; wherein the formation of the trappedpolymerase complex provides information about the identity of the nextbase on the nucleic acid template.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, wherein thetemplate nucleic acid is provided on a nanostructure. Optionally, thesystem includes one or more reagents and instructions necessary to bindtemplate DNA molecules onto a nanostructure. For example, the systemprovides a nanostructure, such as a chip, configured for use withsurface plasmon resonance to determine binding kinetics. An example ofsuch a chip is a CM5 Sensor S chip (GE Healthcare; Piscatawany, N.J.).The system may provide instrumentation such as a surface plasmonresonance instrument. The system may provide streptavidin and/or biotin.Optionally, the system provides biotin-DNA, DNA ligase, buffers, and/orDNA polymerase for preparation of biotinylated template DNA. Optionally,the system provides a gel or reagents (e.g., phenol:chloroform) forbiotinylated DNA purification. Alternatively, the system providesreagents for biotinylated template DNA characterization, for example,mass spectrometry or HPLC. Optionally, the system includes streptavidin,a chip, reagents, instrumentation, and/or instructions forimmobilization of streptavidin on a chip. Optionally, a chip is providedin the system already configured for template DNA coating, wherein thechip is immobilized with a reagent capable of binding template nucleicacids or modified template nucleic acids (e.g., biotinylated templateDNA). Optionally, the system provides reagents for chip regeneration.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, wherein thetemplate nucleic acid is provided on a nanoparticle. Optionally, thesystem includes one or more reagents and instructions necessary to bindtemplate DNA molecules onto a nanoparticle. The nanoparticle may beconfigured for the electrochemical detection of nucleic acid-polymeraseinteraction, for instance, by using gold nanoparticles. Optionally, theDNA-nanoparticle conjugates are formed between aqueous gold colloidsolutions and template DNA molecules including, for example, free thiolor disulfide groups at their ends. The conjugates may include samenucleic acid sequence. Optionally, the nanoparticle conjugates arestabilized against flocculation and precipitation at high temperature(e.g., greater than 60° C.) and high ionic strength (e.g., 1M Na⁺).Optionally, the system provides reagents for preparing template DNAmolecules for nanoparticle attachment, including, generating templateDNA molecules with disulfides or thiols. Disulfide-containing templatenucleic acids may be synthesized using, for example, a 3′-thiol modifiercontrolled-pore glass (CPG) or by beginning with a universal support CPGand adding a disulfide modifier phosphoramidite as the first monomer inthe sequence. The system may provide nucleic acid synthesis reagentsand/or instructions for obtaining disulfide-modified template nucleicacids. Thiol-containing template nucleic acids may also be generatedduring nucleic acid synthesis with a 5′-tritylthiol modifierphosphoramidite. The system may provide reagents and/or instructions fornanoparticle conjugate purification using for example, electrophoresisor centrifugation. Optionally, nanoparticle conjugates are used tomonitor polymerase-template nucleic acid interactions colorimetrically.In this instance, the melting temperature of the nanoparticle conjugateincreases in the presence of strong polymerase binding. Therefore, thestrength of DNA binding can be determined by the change in this meltingtransition, which is observable by a color change. The systemsoptionally include reagents and equipment for detection of the meltingtransition.

Also provided is a system for performing one or more steps of anysequencing method disclosed herein. Optionally, the system includescomponents and reagents necessary to perform a polymerase and templatenucleic acid binding assay in the presence of nucleotides, using adetectable polymerase. Optionally, the polymerase is detectably labeled.Optionally, the polymerase is detected using intrinsic properties of thepolymerase, for example, aromatic amino acids. Optionally, thepolymerase and template nucleic acids present in the system areconfigured for use in solution, without conjugation to a support. Thedetectable label on the polymerase may be a fluorophore, whereinfluorescence is used to monitor polymerase-template nucleic acid bindingevents. Optionally, the detectable polymerase may be used in combinationwith template nucleic acids in solution, or template nucleic acidsconjugated to a support structure. Optionally, one or more cysteineresidues of the polymerase is labeled with Cy3-maleimide. Optionally,the system includes reagents and/or instructions necessary to preparefluorescently labeled polymerase molecules. The system may includereagents and/or instructions for purification of fluorescently labeledpolymerases.

Enhancing Nucleotide Identification Using a Plurality of Nucleotides inMultiple Examination Steps

The disclosed sequencing-by-binding technique can be performed usingmore than one nucleotide during each cycle of an examination step. Forexample, a single examination step optionally can be conducted usingtwo, three, or even four different nucleotides. Optionally, each of thenucleotides is an unlabeled nucleotide, such as a native nucleotide(i.e., dATP, dGTP, dCTP, dTTP or dUTP). Preferably, a primed templatenucleic acid molecule is contacted with a plurality of reaction mixturesin a serial fashion, without incorporation of any nucleotide into theprimed template nucleic acid. Optionally, each different reactionmixture includes a polymerase and a different combination of two orthree different nucleotides. For example, there can be four differentreaction mixtures where, in aggregate, each different nucleotide (e.g.,dATP, dGTP, dCTP, and dTTP) is present two times. This could beaccomplished, for example, by using the following four combinations ofnucleotides: (dATP and dTTP), (dATP and dGTP), (dTTP and dCTP), and(dGTP and dCTP). An alternative would be the combinations: (dGTP anddCTP), (dGTP and dTTP), (dATP and dCTP), and (dATP and dTTP). Yetanother alternative would be the combinations: (dATP and dGTP), (dATPand dCTP), (dGTP and dTTP), and (dCTP and dTTP). Examination steps canbe conducted using four combinations of two different nucleotides, oneafter the other (i.e., such that the first combination is replaced bythe second combination, the second replaced by the third, and the thirdreplaced by the fourth). When this is the case, and when monitoring ofthe interaction of the polymerase with the primed template nucleic acidyields a signal confirming the binding interaction, the next correctnucleotide can be identified as the nucleotide common to two differentnucleotide combinations yielding positive binding signals. If it isdesired to represent each different nucleotide three times among thecollection of nucleotide combinations, an exemplary combination couldbe: (dATP and dTTP), (dATP and dGTP), (dATP and dCTP), (dTTP and dGTP),(dTTP and dCTP), and (dGTP and dCTP). Examination steps can be conductedusing six combinations of two different nucleotides, one after the other(i.e., such that the first combination is replaced by the secondcombination, the second replaced by the third, the third replaced by thefourth, the fourth replaced by the fifth, and the fifth replaced by thesixth). When this is the case, and when monitoring of the interaction ofthe polymerase with the primed template nucleic acid yields a signalconfirming the binding interaction, the next correct nucleotide can beidentified as the nucleotide common to three different nucleotidecombinations yielding a positive binding signal.

One advantage underlying use of more than one nucleotide during theexamination step relates to confirmatory evidence that can be used forestablishing the template sequence in the sequencing-by-bindingprocedure. When, for one or another reason, a single particularexamination step yields only a moderate signal representing the bindinginteraction, testing carried out using the same nucleotide in more thanone combination of nucleotides offers the opportunity for detecting thebinding interaction more than once for each particular nucleotide. Thisenhances correct base calling by reducing the incidence of erroneouslylow, or false-negative results in the monitoring step.

Signal Processing Alternatives

One aspect of the disclosed technique involves identifying two of fourpossible nucleotides that are associated with the highest magnitude ofternary complex formation in an examination step. This can involvegathering and quantifying binding signals by any approach, and thencomparing the results. For example, results from measurement of bindingsignals can be quantified simply by determining peak heights, where thedifferent peaks reflect the extent of binding or interaction of thedifferent components of the binding reaction. Of course, any standardadjustment, such as baseline subtraction, can be applied to this type ofassessment. Another quantitative approach can involve integrating thearea under a binding curve. The integrated values can be compared toidentify the two highest values. Yet another quantitative approach caninvolve fitting a curve to the measured binding signal, and thenprojecting the curve forward to assess the point at which a plateau isreached (e.g., the point at which the first derivative of the curvereaches zero). Of course, other approaches for processing measuredbinding signals also can be applied to quantify binding signals, tocompare the binding signals, and to identify in rank order the twohighest binding signals out of four measurements (i.e., achieved usingfour different nucleotides). Other parameters that may be used forassessing the magnitude of ternary complex formation include peak heightand/or shape of the binding curve. Still other useful parameters includecalculated kinetic parameters, such as time to reach a plateau ofsaturation, k_(obs) (i.e., the rate of ternary complex formation), orR_(eq) (i.e., the maximum magnitude of the projected saturation level,where the projection involves mathematical curve fitting).

Procedural Features of the Methods

Following the examination step, where identities of the next two baseshave been identified via formation of detectable complexes, the reactionconditions may be reset, recharged, or modified as appropriate, inpreparation for the optional incorporation step or an additionalexamination step. Optionally, the identity of the next base has beenidentified without chemically incorporating a nucleotide. Optionally,the identity of the next base is identified with chemical incorporationof a nucleotide, wherein a subsequent nucleotide incorporation has beeninhibited. Optionally, all components of the examination step, excludingthe template nucleic acid being sequenced, are removed or washed away,returning the system to the pre-examination condition. Optionally,partial components of the examination step are removed. Optionally,additional components are added to the examination step.

Optionally, reversible terminator nucleotides are used in theincorporation step to ensure one, and only one nucleotide isincorporated per cycle. No labels are required on the reversibleterminator nucleotides as the base identity is known from theexamination step. Non-fluorescently labeled reversible terminators arereadily available from commercial suppliers. Non-labeled reversibleterminator nucleotides are expected to have faster incorporationkinetics compared to labeled reversible terminators due to their smallersteric footprint, and similar size to natural nucleotides.

Disclosed herein, in part, are reagent cycling sequencing methods,wherein sequencing reagents are introduced, one after another, for everycycle of examination and/or incorporation. Optionally, the sequencingreaction mixture includes a polymerase, a primed template nucleic acid,and at least one type of nucleotide. Optionally, the nucleotide and/orpolymerase are introduced cyclically to the sequencing reaction mixture.Optionally, the sequencing reaction mixture includes a plurality ofpolymerases, primed template nucleic acids, and nucleotides. Optionally,a plurality of nucleotides and/or a plurality of polymerases areintroduced cyclically to the sequencing reaction mixture. Optionally,the examination step of the sequencing reaction has a differentcomposition than the incorporation step of the sequencing reaction.

Optionally, one or more nucleotides are sequentially added to andremoved from the sequencing reaction. Optionally, 1, 2, 3, 4, or moretypes of nucleotides are added to and removed from the reaction mixture.For example, one type of nucleotide is added to the sequencing reaction,removed, and replaced by another type of nucleotide. Optionally, anucleotide type present during the examination step is different from anucleotide type present during the incorporation step. Optionally, anucleotide type present during one examination step is different from anucleotide type present during a sequential examination step (i.e., thesequential examination step is performed prior to an incorporationstep). Optionally, 1, 2, 3, 4 or more types of nucleotides are presentin the examination reaction mixture and 1, 2, 3, 4, or more types ofnucleotides are present in the incorporation reaction mixture.

Optionally, a polymerase is cyclically added to and removed from thesequencing reaction. One or more different types of polymerases may becyclically added to and removed from the sequencing reaction.Optionally, a polymerase type present during the examination step isdifferent from a polymerase type present during the incorporation step.A polymerase type present during one examination step may be differentfrom a polymerase type present during a sequential examination step(i.e., the sequential examination step is performed prior to anincorporation step).

Optionally, conditions such as the presence of reagents, pH,temperature, and ionic strength are varied throughout the sequencingreaction. Optionally, a metal is cyclically added to and removed fromthe sequencing reaction. For example, a catalytic metal ion may beabsent during an examination step and present during an incorporationstep. Alternatively, a polymerase inhibitor may be present during anexamination step and absent during an incorporation step. Optionally,reaction components that are consumed during the sequencing reaction aresupplemented with the addition of new components at any point during thesequencing reaction.

Nucleotides can be added one type at a time, with the polymerase, to areaction condition that favors closed-complex formation. The polymerasebinds only to the template nucleic acid if the next correct nucleotideis present. A wash step after every nucleotide addition ensures allexcess polymerases and nucleotides not involved in a closed-complex areremoved from the reaction mixture. Optionally, the wash step removes anybound polymerase and nucleotide. If the nucleotides are added one at atime, in a known order, the next base on the template nucleic acid isdetermined by the formation of a closed-complex when the addednucleotide is the next correct nucleotide. The closed-complex may beidentified by both the conformational change and the increased stabilityof the polymerase-template nucleic acid-nucleotide interaction.Optionally, the stability of the closed-complex formed in the presenceof the next correct nucleotide is at least an order of magnitude greaterthan the unstable interactions of the polymerase with the templatenucleic acid in the presence of incorrect nucleotides. The use of a washstep ensures that there are no unbound nucleotides and polymerases andthat the only nucleotides present in the reaction are those sequesteredin a closed-complex with a polymerase and a template nucleic acid. Oncethe next base on the template nucleic acid is determined, the nextcorrect nucleotide sequestered in the closed complex may be incorporatedby flowing in reaction conditions that favor dissociation ordestabilization of the closed-complex and extending the template nucleicacid primer strand by one base (incorporation). Therefore, the wash stepensures that the only nucleotide incorporated is the next correctnucleotide from the closed-complex. This reagent cycling method may berepeated and the nucleic acid sequence determined. This reagent cyclingmethod may be applied to a single template nucleic acid molecule, or tocollections of clonal populations such as PCR products or rolling-circleamplified DNA. Many different templates can be sequenced in parallel ifthey are arrayed, for instance, on a solid support. Optionally, the washstep destabilizes binary complex formation. Optionally, the washing isperformed for a duration of time that ensures that the binary complex isremoved, leaving the stabilized closed complex in the reaction mixture.Optionally, the wash step includes washing the reaction with a highionic strength or a high pH solution. Optionally, the wash stepdestabilizes both binary and ternary complexes, removing any boundpolymerase and nucleotide from an immobilized primed template nucleicacid molecule (optionally blocked at its 3′-end from forming aphosphodiester bond). Such a wash step may involve contactingimmobilized complexes with a solution that includes EDTA to chelatemetal ions.

Optionally, the incorporation step is a three stage process. In thefirst stage, all four nucleotide types are introduced into a reactionincluding a primed template nucleic acid, with a high fidelitypolymerase, in reaction conditions which favor the formation of aclosed-complex, and the next correct nucleotides are allowed to formstable closed-complexes with the template nucleic acid. In a secondstage, excess nucleotides and unbound polymerase are washed away. In athird stage, reaction conditions are modified so that the closed-complexis destabilized and the sequestered nucleotides within theclosed-complex become incorporated into the 3′-end of the templatenucleic acid primer. In an alternative approach, the second stage ismodified to remove completely any of the high fidelity polymerase andcognate nucleotide that may have been present in the closed-complex, andthe removed components are then replaced with a second polymerase andone or more nucleotides (e.g., reversible terminator nucleotides).Formation of tight polymerase-nucleic acid complexes in theincorporation step can be enabled by standard techniques such as fusinga non-specific DNA binding domain to the polymerase (e.g., the Phusionpolymerase, which is available from Thermo Fisher Scientific; Waltham,Mass.), and utilizing high concentrations of nucleotides to ensurecorrect nucleotides are always present in the closed complex.

Polymerase molecules bind to primed template nucleic acid molecules in afingers-closed conformation in the presence of the next correctnucleotide even in the absence of divalent metal ions that are typicallyrequired for polymerase synthesis reactions. The conformational changetraps the nucleotide complementary to the next template base within theactive site of the polymerase. Optionally, the formation of theclosed-complex may be used to determine the identity of next base on thetemplate nucleic acid. Optionally, the primed template nucleic acids maybe contacted serially by different nucleotides in the presence ofpolymerase, in the absence of catalytic divalent metal ions; wherein theformation of a closed complex indicates the nucleotide currently incontact with the template nucleic acid is the complementary nucleotideto the next base on the nucleic acid. A known order of nucleotides (inthe presence of polymerase and absence of catalytic metal ions) broughtinto contact with the template nucleic acid ensures facileidentification of the complementary nucleotide based on the particularposition in the order that induces closed-complex formation. Optionally,an appropriate wash step may be performed after every nucleotideaddition to ensure removal of all excess enzymes and nucleotides,leaving behind only the polymerase that is bound to nucleic acids in aclosed-complex with the next correct nucleotide at the active site. Theclosed-complex may be identified by means that reveal the conformationalchange of the polymerase in the closed conformation or by means thatreveal the increased stability of thepolymerase/nucleic-acid/next-correct-nucleotide complex compared tobinary polymerase-nucleic acid complexes or compared to unstableinteractions between the polymerase, primed template nucleic acid andincorrect nucleotides.

Optionally, the process of identifying the next complementary nucleotide(examination step) includes the steps of contacting immobilized primedtemplate nucleic acids with an examination mixture including polymeraseand nucleotides of one kind under conditions that inhibit the chemicalincorporation of the nucleotide, removing unbound reagents by a washstep, detecting the presence or absence of polymerase closed-complex onthe immobilized nucleic acids, and repeating these steps serially, withnucleotides of different kinds until a closed-complex formation isdetected. The closed-complex may be identified by both theconformational change and the increased stability of thepolymerase/nucleic-acid/next-correct-nucleotide complex. The wash stepbetween successive nucleotide additions may be eliminated by the use ofdetection mechanisms that can detect the formation of the closed complexwith high fidelity, for instance, evanescent wave sensing methods ormethods that selectively monitor signals from the closed-complex. Theexamination steps noted above may be followed by an incorporation stepincluding, contacting the closed-complex with catalytic metal ions tocovalently add the nucleotide sequestered in the closed-complex to the3′-end of the primer. Optionally, the incorporation step may include,contacting the immobilized nucleic acids with a pre-incorporationmixture including a combination of multiple types of nucleotides andpolymerase under conditions that inhibit the chemical incorporation ofthe nucleotides; wherein the pre-incorporation mixture may containadditives and solution conditions to ensure highly efficientclosed-complex formation (e.g., low-salt conditions). The methods mayalso include performing a wash step to remove unbound reagents andproviding the immobilized complexes with an incorporation mixture,including catalytic metal ions, to chemically incorporate nucleotidessequestered within the active site of the polymerase. Thepre-incorporation mixture ensures highly efficient closed-complexformation, while the wash step and incorporation mixture ensure theaddition of a single nucleotide to the 3′-end of the primer. Optionally,the incorporation step may occur directly after examination an additionof one type of nucleotide. For instance, a repeated pattern used forsequencing may include the following flow pattern (i) dATP+/polymerase,(ii) Wash, (iii) Mg²⁺, (iv) Wash, (v) dTTP+/polymerase, (vi) Wash, (vii)Mg²⁺, (viii) Wash, (ix) dCTP+/polymerase, (x) Wash (xi) Mg²⁺, (xii)Wash, (xiii) dGTP+/polymerase, (xiv) Wash, (xv) Mg²⁺, (xvi) Wash.Optionally, the repeated pattern used for sequencing may include (i)dATP+/polymerase, (ii) Wash, (iii) dTTP+/polymerase, (iv) Wash, (v)dGTP+/polymerase, (vi) Wash, (vii) dCTP+/polymerase, (viii) Wash, (ix)Pre-incorporation mixture, (x) Wash, (xi) Mg, (xii) Wash. The wash stepstypically contain metal ion chelators and other small molecules toprevent accidental incorporations during the examination steps. Afterthe incorporation step, the primer strand is typically extended by onebase. Repeating this process, sequential nucleobases of a nucleic acidmay be identified, effectively determining the nucleic acid sequence.Optionally, the examination step is performed at high salt conditions,for example, under conditions of 50 mM to 1,500 mM salt.

For sequencing applications, it can be advantageous to minimize oreliminate fluidics and reagents exchange. Removing pumps, valves andreagent containers can allow for simplified manufacturing of smallerdevices. Disclosed herein, in part, are “all-in” sequencing methods,wherein the need to introduce reagents one after another, for everycycle of examination and/or incorporation, is eliminated. Reagents areadded only once to the reaction, and sequencing-by-synthesis isperformed by manipulating reagents already enclosed within thesequencing reaction. A scheme such as this requires a method todistinguish different nucleotides, a method to synchronize incorporationof nucleotides across a clonal population of nucleic acids and/or acrossdifferent nucleic acid molecules, and a method to ensure only onenucleotide is added per cycle.

Optionally, the sequencing reaction mixture includes a polymerase, aprimed template nucleic acid, and at least one type of nucleotide.Optionally, the sequencing reaction mixture includes a plurality ofpolymerases, primed template nucleic acids, and nucleotides. As providedherein, a polymerase refers to a single polymerase or a plurality ofpolymerases. As provided herein, a primed template nucleic acid ortemplate nucleic acid refers to a single primed template nucleic acid orsingle template nucleic acid, or a plurality of primed template nucleicacids or a plurality of template nucleic acids. As provided herein, anucleotide refers to one nucleotide or a plurality of nucleotides. Asprovided herein, a single nucleotide is one nucleotide. Optionally, thesequencing reaction nucleotides include, but are not limited to, 1, 2,3, or 4 of the following nucleotides: dATP, dGTP, dCTP, dTTP, and dUTP.

Optionally, the examination step and the incorporation step take placein a single sequencing reaction mixture.

Optionally, 1, 2, 3, 4 or more types of nucleotides (e.g., dATP, dGTP,dCTP, dTTP) are present in the reaction mixture together at the sametime, wherein one type of nucleotide is a next correct nucleotide. Thereaction mixture further includes at least one polymerase and at leastone primed template nucleic acids. Optionally, the template nucleic acidis a clonal population of template nucleic acids. Optionally, thepolymerase, primed template nucleic acid, and the nucleotide form aclosed-complex under examination reaction conditions.

In the provided methods, four types of nucleotides can be present atdistinct and different concentrations wherein the diffusion and bindingtimes of the polymerase to the template nucleic acid are different foreach of the four nucleotides, should they be the next correctnucleotide, due to the different concentrations of the four nucleotides.For example, the nucleotide at the highest concentration would bind toits complementary base on the template nucleic acid at a fast time, andthe nucleotide at the lowest concentration would bind to itscomplementary base on the template nucleic acid at a slower time;wherein binding to the complementary base on the template nucleic acidrefers to the polymerase binding to the template nucleic acid with thenext correct nucleotide in a closed closed-complex. The identity of thenext correct nucleotide is therefore determined by monitoring the rateor time of binding of polymerase to the template nucleic acid in aclosed-complex. Optionally, the four types of nucleotides may bedistinguished by their concentration, wherein the differentconcentrations of the nucleotides result in measurably differenton-rates for the polymerase binding to the nucleic acid. Optionally, thefour types of nucleotides may be distinguished by their concentration,wherein the different concentrations of the nucleotides result inmeasurably different on-rates for the formation of a stabilizedclosed-complex.

Optionally, the polymerase is labeled. In some instances, the polymeraseis not labeled (i.e., does not harbor an exogenous label, such as afluorescent label) and any label-free detection method disclosed hereinor known in the art is employed. Optionally, the binding of thepolymerase to the nucleic acid is monitored via a detectable feature ofthe polymerase. Optionally, the formation of a stabilized closed-complexis monitored via a detectable feature of the polymerase. A detectablefeature of the polymerase may include, but is not limited to, optical,electrical, thermal, colorimetric, mass, and any combination thereof.

Optionally, 1, 2, 3, 4, or more nucleotides types (e.g., dATP, dTTP,dCTP, dGTP) are tethered to 1, 2, 3, 4, or more different polymerases;wherein each nucleotide type is tethered to a different polymerase andeach polymerase has a different exogenous label or a detectable featurefrom the other polymerases to enable its identification. All tetherednucleotide types can be added together to a sequencing reaction mixtureforming a closed-complex including a tethered nucleotide-polymerase; theclosed-complex is monitored to identify the polymerase, therebyidentifying the next correct nucleotide to which the polymerase istethered. The tethering may occur at the gamma phosphate of thenucleotide through a multi-phosphate group and a linker molecule. Suchgamma-phosphate linking methods are standard in the art, where afluorophore is attached to the gamma phosphate linker. Optionally,different nucleotide types are identified by distinguishable exogenouslabels. Optionally, the distinguishable exogenous labels are attached tothe gamma phosphate position of each nucleotide.

Optionally, the sequencing reaction mixture includes a catalytic metalion. Optionally, the catalytic metal ion is available to react with apolymerase at any point in the sequencing reaction in a transientmanner. To ensure robust sequencing, the catalytic metal ion isavailable for a brief period of time, allowing for a single nucleotidecomplementary to the next base in the template nucleic acid to beincorporated into the 3′-end of the primer during an incorporation step.In this instance, no other nucleotides, for example, the nucleotidescomplementary to the bases downstream of the next base in the templatenucleic acid, are incorporated. Optionally, the catalytic metal ionmagnesium is present as a photocaged complex (e.g., DM-Nitrophen) in thesequencing reaction mixture such that localized UV illumination releasesthe magnesium, making it available to the polymerase for nucleotideincorporation. Furthermore, the sequencing reaction mixture may containEDTA, wherein the magnesium is released from the polymerase active siteafter catalytic nucleotide incorporation and captured by the EDTA in thesequencing reaction mixture, thereby rendering magnesium incapable ofcatalyzing a subsequent nucleotide incorporation.

Thus, in the provided methods, a catalytic metal ion can be present in asequencing reaction in a chelated or caged form from which it can bereleased by a trigger. For example, the catalytic metal ion catalyzesthe incorporation of the closed-complex next correct nucleotide, and, asthe catalytic metal ion is released from the active site, it issequestered by a second chelating or caging agent, disabling the metalion from catalyzing a subsequent incorporation. The localized release ofthe catalytic metal ion from its cheating or caged complex is ensured byusing a localized uncaging or un-chelating scheme, such as an evanescentwave illumination or a structured illumination. Controlled release ofthe catalytic metal ions may occur for example, by thermal means.Controlled release of the catalytic metal ions from their photocagedcomplex may be released locally near the template nucleic acid byconfined optical fields, for instance by evanescent illumination such aswaveguides or total internal reflection microscopy. Controlled releaseof the catalytic metal ions may occur for example, by altering the pH ofthe solution near the vicinity of the template nucleic acid. Chelatingagents such as EDTA and EGTA are pH dependent. At a pH below 5, divalentcations Mg²⁺ and Mn²⁺ are not effectively chelated by EDTA. A method tocontrollably manipulate the pH near the template nucleic acid allows thecontrolled release of a catalytic metal ion from a chelating agent.Optionally, the local pH change is induced by applying a voltage to thesurface to which the nucleic acid is attached. The pH method offers anadvantage in that that metal goes back to its chelated form when the pHis reverted back to the chelating range.

Optionally, a catalytic metal ion is strongly bound to the active siteof the polymerase, making it necessary to remove the polymerase from thetemplate nucleic acid after a single nucleotide incorporation. Theremoval of polymerase may be accomplished by the use of a highlydistributive polymerase, which falls off the 3′-end of the strand beingsynthesized (e.g., primer) after the addition of every nucleotide. Theunbound polymerase may further be subjected to an electric or magneticfield to remove it from the vicinity of the nucleic acid molecules. Anymetal ions bound to the polymerase may be sequestered by chelatingagents present in the sequencing reaction mixture, or by molecules whichcompete with the metal ions for binding to the active site of thepolymerase without disturbing the formation of the closed complex. Theforces which remove or move the polymerase away from the templatenucleic acid (e.g., electric field, magnetic field, and/or chelatingagent) may be terminated, allowing for the polymerase to approach thetemplate nucleic acid for another round of sequencing (i.e., examinationand incorporation). The next round of sequencing, in a non-limitingexample, includes the formation of a closed-complex, removing unboundpolymerase away from the vicinity of the template nucleic acid and/orclosed-complex, controlling the release of a catalytic metal ion toincorporate a single nucleotide sequestered within the closed-complex,removing the polymerase which dissociates from the template nucleic acidafter single incorporation away from the vicinity of the templatenucleic acid, sequestering any free catalytic metal ions through the useof chelating agents or competitive binders, and allowing the polymeraseto approach the template nucleic acid to perform the next cycle ofsequencing.

Described above are polymerase-nucleic acid binding reactions for theidentification of a nucleic acid sequence. However, nucleic acidsequence identification may include information regarding nucleic acidmodifications, including methylation and hydroxymethylation. Methylationmay occur on cytosine bases of a template nucleic acid. DNA methylationmay stably alter the expression of genes. DNA methylation is alsoindicated in the development of various types of cancers,atherosclerosis, and aging. DNA methylation therefore can serve as anepigenetic biomarker for human disease.

Optionally, one or more cytosine methylations on a template nucleic acidare identified during the sequencing by binding methods provided herein.The template nucleic acid may be clonally amplified prior to sequencing,wherein the amplicons include the same methylation as their templatenucleic acid. Amplification of the template nucleic acids may includethe use of DNA methyltransferases to achieve amplicon methylation. Thetemplate nucleic acids or amplified template nucleic acids are providedto a reaction mixture including a polymerase and one or more nucleotidetypes, wherein the interaction between the polymerase and nucleic acidsis monitored. Optionally, the interaction between the polymerase andtemplate nucleic acid in the presence of a methylated cytosine isdifferent than the interaction in the presence of an unmodifiedcytosine. Therefore, based on examination of a polymerase-nucleic acidinteraction, the identity of a modified nucleotide is determined.

Optionally, following one or more examination and/or incorporationsteps, a subset of nucleotides is added to reduce or reset phasing.Thus, the methods can include one or more steps of contacting a templatenucleic acid molecule being sequenced with a composition comprising asubset of nucleotides and an enzyme for incorporating the nucleotidesinto the strand opposite the template strand of the nucleic acidmolecule. The contacting can occur under conditions to reduce phasing inthe nucleic acid molecule. Optionally, the step of contacting thetemplate nucleic acid molecule occurs after an incorporation step and/orafter an examination step. Optionally, the contacting occurs after 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75,80, 85, 90, 95, or 100 rounds or more of sequencing, i.e., rounds ofexamination and incorporation. Optionally, the contacting occurs after30 to 60 rounds of sequencing. Optionally, the contacting occurs afterevery round of sequencing (i.e., after one set of examination andincorporation steps). Optionally, multiple contacting steps occur afterevery round of sequencing, wherein each contacting step may comprisedifferent subsets of nucleotides. Optionally, the method furthercomprises one or more washing steps after contacting. Optionally, thesubset comprises two or three nucleotides. Optionally, the subsetcomprises three nucleotides. Optionally, the subset of nucleotides isselected from three of dATP, dGTP, dCTP, dTTP or a derivative thereof.Optionally, the three nucleotides comprise adenosine, cytosine, andguanine. Optionally, the three nucleotides comprise adenosine, cytosine,and thymine. Optionally, the three nucleotides comprise cytosine,guanine and thymine. Optionally, the three nucleotides compriseadenosine, guanine and thymine. Optionally, each round of contactingcomprises the same subset or different subsets of nucleotides.Optionally, sequencing of a nucleic acid template is monitored and thecontacting with the subset of nucleotides occurs upon detection ofphasing. See also for example, U.S. Pat. No. 8,236,532, which isincorporated herein by reference in its entirety.

Optionally, the sequencing reaction involves a plurality of templatenucleic acids, polymerases and/or nucleotides, wherein a plurality ofclosed-complexes is monitored. Clonally amplified template nucleic acidsmay be sequenced together wherein the clones are localized in closeproximity to allow for enhanced monitoring during sequencing.Optionally, the formation of a closed-complex ensures the synchronicityof base extension across a plurality of clonally amplified templatenucleic acids. The synchronicity of base extension allows for theaddition of only one base per sequencing cycle.

EXAMPLES

The following Example illustrates examination steps that employedcatalytic amounts of Mg²⁺ ion in combination with a primer having a3′-blocking group. The blocking group of the primer was removed aftermeasuring binding of the primed template nucleic acid molecule topolymerase in the presence of each different native nucleotide. Themeasured binding was sufficient to identify which of the nucleotidesrepresented the cognate nucleotide for a particular position. In thenext step of the workflow, a reversible terminator nucleotide wasincorporated without any intervening examination step (i.e., withoutintervening binding, detection or identification of any nucleotide).Notably, examination and incorporation steps were carried out using twodifferent polymerase enzymes. Optionally, a single polymerase, such asthe 3PDX polymerase disclosed in U.S. Pat. No. 8,703,461 for the purposeof interrogating nucleotide analogs and incorporating reversibleterminator nucleotides, also may be used.

Example 1 describes examination of a primed template nucleic acidmolecule, where the primer strand was blocked from extension at its3′-end. The examination step (i.e., involving measuring interactionbetween the primed template nucleic acid molecule, the polymerase, and atest nucleotide) was conducted in the presence of a catalytic metal ionwith the intention of enhancing discriminatory activity of thepolymerase enzyme. Results presented below demonstrated efficientidentification of the next correct nucleotide, and even the followingnext correct nucleotide.

Example 1 Examination of a Primed Template Nucleic Acid Molecule Havinga 3′-Blocked Primer in the Presence of Catalytic Concentrations ofMagnesium Ion

A FORTEBIO® (Menlo Park, Calif.) OCTET® instrument employing biolayerinterferometry to measure binding reactions at the surface of a fiberoptic tip was used in a multiwell plate format to illustrate thesequencing technique. Template strands biotinylated at their 5′-endswere used to immobilize primed template nucleic acid molecule onto fiberoptic tips functionalized with streptavidin (SA) according to standardprocedures. A 3′-blocked primer was prepared by incorporating a cognatereversible terminator nucleotide that included a 3′-ONH₂ blocking group,using Therminator DNA polymerase (New England BioLabs Inc.; Ipswich,Mass.) according to the manufacturer's instructions. A description ofthe reversible terminator nucleotide can be found in U.S. Pat. No.7,544,794, the disclosure of which is incorporated by reference. Bindingof incoming nucleotides was investigated using 68 units/mL of Bsu DNApolymerase (New England BioLabs Inc.; Ipswich, Mass.) in a buffer thatfurther included 30 mM Tris (pH 8.0), 220 mM KCl, 160 mM potassiumglutamate, either 0.1 mM or 1 mM MgCl₂, 0.01% Tween-20, and 1 mMβ-mercaptoethanol. Ternary complex formation indicating cognatenucleotide binding was investigated by contacting the primed templatenucleic acid molecule having the 3′-blocked primer with the Bsu DNApolymerase and one of four native dNTP nucleotides (dATP, dGTP, dCTP,and dTTP) for a period of 20 seconds in a serial fashion. Each of thedifferent nucleotides was used at a concentration of 100 μM during theexamination procedure. Thereafter, biosensors were washed with asolution that included 20 mM EDTA for 25 seconds to chelate magnesiumions. The biosensors were then equilibrated with regeneration bufferthat included 30 mM Tris (pH 8.0), 220 mM KCl, 160 mM potassiumglutamate, 1 mM MgCl₂, 0.01% Tween-20, 1 mM β-mercaptoethanol. The samesteps were repeated for the remaining nucleotides in sequence untilcollecting all binding curves for all four dNTPs. After completingexamination of the different nucleotides, and acquiring measurement datafor identifying the next correct nucleotide, the biosensor wastransferred into a cleavage buffer solution (1 M sodium acetate pH 5.5and 500 mM NaNO₂) for 60 seconds to remove the blocking group from the3′-end of the primer. Biosensors were next equilibrated with aregeneration buffer (20 mM Tris pH 8.0, 10 mM KCl, and 0.01% Tween-20).Correct nucleotide was subsequently incorporated using the Therminatorpolymerase at a concentration of 30 units/mL in a buffer that included20 mM Tris (pH 8.8), 10 mM ammonium sulfate, 10 mM KCl, 2 mM MgCl₂, 0.1%Triton-X-100, and all four reversible terminator nucleotides at aconcentration of 100 μM each. All buffers were prepared with HPLC gradewater and the incorporation buffer included HPLC water with 0.5 wt %OH—NH₂. The incorporation step was carried out for 60 seconds, afterwhich time the bound polymerase was washed away from the biosensor with20 mM EDTA for 5 seconds before commencing the next examination cycle,as described above.

FIGS. 1A-1B show the traces for independent binding of all fournucleotides followed by the cleavage traces and incorporation traces, asdiscussed above. The expected base sequence in this example was GAC. Asdescribed above, a 3′-ONH₂ blocked primer was first formed byincorporating a reversible terminator using the Therminator polymerase.Next, for each cycle of examination the blocked primed template nucleicacid molecule was contacted with polymerase and a different nucleotide(dATP or dTTP or dGTP or dCTP) in the presence of catalytic magnesiumions for 20 seconds. High binding signals were observed if the examinednucleotide included the complementary base to the next base of thetemplate strand. In addition to this peak, a second-high binding signalwas also observed for the second correct base complementary to thesecond next base of the template strand. After all four nucleotides hadbeen examined, a cleavage reaction removed the 3′ blocking group fromthe primer. After removing the cleavage reagent with two wash steps(corresponding to the two steps with progressively reduced bindingsignals immediately following the cleavage step), a single incorporationreaction was carried out to add the next reversible terminatornucleotide. The procedure can be used for identifying the next correctnucleotide (next incoming nucleotide at the n+1 position), and can berepeated a plurality of times to determine the sequence of the templatenucleic acid. As well, the results showed how the correct nucleotide atthe n+2 position also could be determined. This observation wasreproduced for all the positions in the sequence. Optionally, serialincorporation of two reversible terminators can be carried out withoutintervening examination steps using different types of nucleotides(i.e., other than reversible terminators) to speed the process ofsequence determination.

Example 2 describes a collection of procedures that confirmed the nexttwo correct nucleotides could be identified from a single complete cycleof examination reactions in a sequencing-by-binding protocol. Morespecifically, the following Example further illustrates how the nextcorrect nucleotide, and the subsequent correct nucleotide wereidentified from one cycle of examining four nucleotides in a serialfashion, and then comparing the results. The use of reversibleterminator nucleotides ensured that only a single nucleotide wasincorporated at the end of the examination cycle.

Example 2 Examination of a Primed Template Nucleic Acid Molecule toIdentify the Next Two Correct Nucleotides

A FORTEBIO® (Menlo Park, Calif.) OCTET® instrument employing biolayerinterferometry to measure binding reactions at the surface of a fiberoptic tip was used in a multiwell plate format to illustrate thesequencing technique. Template strands biotinylated at their 5′-endswere used to immobilize primed template nucleic acid molecule onto fiberoptic tips functionalized with streptavidin (SA) according to standardprocedures. The primed template nucleic acid molecule was contacted for20 seconds with 68 U/mL of Bsu DNA polymerase (New England BioLabs Inc.;Ipswich, Mass.) in a buffer that included 30 mM Tris (pH 8.0), 220 mMKCl, 160 mM potassium glutamate, 2 mM NiSO₄, 0.01% Tween-20, 1 mMβ-mercaptoethanol, and the first incoming test nucleotide (dTTP) at aconcentration of 100 μM. Thereafter, biosensors were washed with asolution that included 20 mM EDTA for 25 seconds to chelate nickel ionsand remove any complexed polymerase. Biosensors were next contacted witha regeneration buffer that included 30 mM Tris (pH 8.0), 220 mM KCl, 160mM potassium glutamate, 2 mM NiSO₄, 0.01% Tween-20, 1 mMβ-mercaptoethanol. The same steps were repeated for the individualremaining nucleotides (dATP, dGTP, and dCTP) in sequence untilcollecting binding data for all four dNTPs. After completing examinationof the different nucleotides, and acquiring measurement data foridentifying the next correct nucleotide, the biosensor was transferredinto a solution that included 20 mM EDTA for 25 seconds to chelatenickel ions and remove any complexed polymerase. The biosensors werethen contacted with a buffer that included 20 mM Tris (pH 8.0), 10 mMKCl, and 0.01% Tween-20. Subsequently, a reversible terminatornucleotide was incorporated using Therminator polymerase (New EnglandBioLabs Inc.; Ipswich, Mass.) at 30 units/mL in a buffer made 20 mM Tris(pH 8.8), 10 mM ammonium sulfate, 10 mM KCl, 2 mM MgCl₂, and 0.1%Triton-X-100, together with all four 3′-ONH₂ derivatives atconcentrations of 100 μM each. All buffers were prepared with HPLC gradewater and the incorporation buffer included HPLC water with 0.5 wt %OH—NH₂. The incorporation step was allowed to proceed for 60 seconds.Bound polymerase was removed from the biosensor by washing with asolution of 20 mM EDTA for 5 seconds before initiating the cleavagereaction. The incorporated reversible terminator moiety was deaminatedfor 60 seconds using a cleavage buffer made 1 M sodium acetate (pH 5.5)and 500 mM NaNO₂. The biosensor was next equilibrated with aregeneration buffer appropriate for the DNA polymerase enzyme of theexamination step before commencing the next cycle. This procedure wasused for examination of primed template nucleic acid molecules havingfree 3′-OH groups.

FIG. 2 presents interferometry traces for serial examinations carriedout using all four nucleotides, along with reversible terminatornucleotide incorporation and cleavage traces. In all instances,examination was conducted using primed template nucleic acid moleculeshaving free 3′-OH groups. The expected sequence was AGTG. The tracesillustrate two high-binding signals, where the signal indicating themost extensive ternary complex formation (e.g., as judged by highestmagnitude or signal trajectory indicating a later plateau) identifiedthe next correct nucleotide. The signal indicating the second mostextensive ternary complex formation (e.g., as judged by the secondhighest magnitude or signal trajectory indicating a later plateau)identified the subsequent next correct nucleotide. Incorporating asingle reversible terminator nucleotide extended the primer by onenucleotide. Taken together, the results presented in FIGS. 1 and 2confirmed that the sequencing-by-binding platform was capable ofidentifying the next two nucleotides downstream of the primer in aprimed template nucleic acid molecule.

The following Example demonstrates double incorporation of reversibleterminator nucleotides, with removal of the reversible terminatormoieties after each of the first and second incorporations. While theprocedure described immediately below involved examination of primedtemplate nucleic acids having free 3′-OH groups and incorporation of tworeversible terminators with removal of the reversible terminator moietybefore examining the next test nucleotide, variations on this procedurecan be used and fall within the scope of the disclosure. For example,the order of reactions can be changed so that reversibly terminatedprimers are used in the examination step, with the reversible terminatormoiety being cleaved from the primer after all examination reactionshave been completed. Another reversible terminator nucleotide can beincorporated, its reversible terminator moiety cleaved, and thenoptionally yet another reversible terminator incorporated to provide aprimer having a blocked 3′-end. The blocked primer can be used in theexamination steps, after which the reversible terminator moiety isremoved, and the next two reversible terminator nucleotidesincorporated.

Example 3 Rapid Sequencing by Double Incorporation of ReversibleTerminator Nucleotides

Procedures for preparing biosensors having immobilized primed templatenucleic acids, for performing examination reactions in the presence ofnon-catalytic metal ions that inhibit polymerase-mediated incorporation,and for performing incorporation and cleavage reactions were essentiallyas described in Example 2. In this instance however, two reversibleterminator nucleotides were incorporated, and their reversibleterminator moieties removed before subsequent examination steps wereconducted using native nucleotides. Magnitudes of monitored bindingsignals indicating complex formation were assessed simply by judgingrelative peak heights. Alternative assessments were based on calculatingareas under the curves, kinetic trajectories leading to plateaus, etc.These approaches for assessing relative magnitudes of binding signalsapply generally to the methods described herein.

FIG. 3 presents interferometry traces for examination carried out usingall four nucleotides, along with incorporation and cleavage tracesobtained by examination of primed template nucleic acid molecules havingfree 3′-OH groups. The expected sequence was TAGT. The traces illustratetwo high-binding signals, where the higher of the two signals identifiedthe next correct nucleotide, and the second-highest binding signalidentified the subsequent correct nucleotide downstream of the primer.Referring to the first set of four examination reactions, the highestbinding signal indicated that the next correct nucleotide was dTTP, andthe second-highest binding signal indicated the subsequent correctnucleotide was dATP. Referring to the second set of four examinationreactions (i.e., following the double incorporation of reversibleterminator nucleotides), the highest binding signal indicated the nextcorrect nucleotide was dGTP, and the second-highest binding signalindicated the subsequent correct nucleotide was dTTP. Incorporating tworeversible terminator nucleotides extended the primer by two nucleotidesfor the next round of examination.

FIG. 4 presents interferometry traces for examination carried out usingall four nucleotides, along with incorporation and cleavage tracesobtained by examination of primed template nucleic acid molecules havingfree 3′-OH groups. The expected sequence was GGC. The traces illustrateonly a single high-binding signal during the examination of dGTP. Thiscorrectly indicated that both of the next two nucleotides were the same(i.e., dGTP). Incorporating two reversible terminator nucleotidesextended the primer by two nucleotides for the next round ofexamination.

Example 4 describes how pairwise combinations of nucleotides could beused during examination steps to increase confidence in the accuracy ofbase calling, and to aid in identification of the next two correctnucleotides for a primed template nucleic acid molecule. All examinationreactions were carried out using nucleotide combinations, as discussedabove. The demonstration included examination of one primed templatenucleic acid molecule having a free 3′-OH, and another having areversibly terminated primer.

Example 4 Examination Using Pairwise Combinations of Nucleotides

A FORTEBIO® (Menlo Park, Calif.) OCTET® instrument employing biolayerinterferometry to measure binding reactions at the surface of a fiberoptic tip was used in a multiwell plate format to illustrate thesequencing technique. Template strands biotinylated at their 5′-endswere used to immobilize primed template nucleic acid molecule onto fiberoptic tips functionalized with streptavidin (SA) according to standardprocedures. The first primed template nucleic acid molecule included afree 3′-OH group on the primer, and was examined in the presence of anon-catalytic metal ion (Ni²⁺) that inhibit polymerase-mediatedincorporation to stabilize ternary complexes. The second primed templatenucleic acid molecule included a reversible terminator moiety (i.e.,3′-ONH₂) on the 3′ terminal nucleotide, and was also examined in thepresence of Ni²⁺, although the non-catalytic metal ion is not believednecessary when using the blocked primer. The immobilized primed templatenucleic acids were contacted for 20 seconds with 68 U/mL of Bsu DNApolymerase (New England BioLabs Inc.; Ipswich, Mass.) in a buffer thatincluded 30 mM Tris (pH 8.0), 220 mM KCl, 160 mM potassium glutamate, 2mM NiSO₄, 0.01% Tween-20, 1 mM β-mercaptoethanol, and the first incomingpair of test nucleotides (dATP and dGTP) at concentrations of 100 μMeach. Thereafter, biosensors were washed with a solution that included20 mM EDTA for 25 seconds to chelate nickel ions and remove anycomplexed polymerase. Biosensors were next contacted with a regenerationbuffer that included 30 mM Tris (pH 8.0), 220 mM KCl, 160 mM potassiumglutamate, 2 mM NiSO₄, 0.01% Tween-20, 1 mM β-mercaptoethanol. The samesteps were repeated for the individual remaining pairs of nucleotidesuntil monitoring data for binding of all four dNTPs (each being presentat 100 μM concentrations) had been collected. The remaining pairs ofnucleotides used in the procedure were: dATP and dCTP; dATP and dTTP;dGTP and dCTP; dTTP and dGTP; and dTTP and dCTP. After completingexamination of the different nucleotide pairs, and acquiring measurementdata, the biosensors were transferred into a solution that included 20mM EDTA for 25 seconds to chelate nickel ions and remove any complexedpolymerase. The biosensors were then contacted with a buffer thatincluded 20 mM Tris (pH 8.0), 10 mM KCl, and 0.01% Tween-20. In the casewhere the primed template nucleic acid already included a 3′-ONH₂reversible terminator moiety, that moiety was cleaved to reveal a free3′-OH group. More specifically, the incorporated reversible terminatormoiety was deaminated for 60 seconds using a cleavage buffer made of 1 Msodium acetate (pH 5.5) and 500 mM NaNO₂. The biosensors were thenavailable to participate in subsequent incorporation reactions.

Results shown in FIGS. 5A-5C, obtained using primers terminating in free3′-OH groups, confirmed that the next correct nucleotide, and thesubsequent correct nucleotide were easily identified by using pairwisecombinations of nucleotides in the examination step. FIG. 5A shows thatthe highest magnitudes of ternary complex formation were associated withnucleotide pairs that shared dATP, thereby indicating that thisnucleotide was the next correct base. Of the three nucleotide pairscontaining the first correct nucleotide (dATP), the “AT” combination(contained dATP and dTTP) yielded the highest magnitude of ternarycomplex formation, as judged by peak height and projected latest time toreach a plateau. Thus, dTTP was the subsequent correct nucleotide.Identity of dTTP as the correct second position was supported by thefact that the TG combination yielded a stronger signal than TC. FIGS.5B-5C graphically present results of parameters calculated by the Octetinterferometry instrument that also could be used for assessing theextent of ternary complex formation. Here the highest R_(eq) values andthe lowest k_(obs) values indicated signal strengths in the bindingassay. Again, each parameter supported determination that the next twocognate nucleotides were AT.

Results shown in FIGS. 6A-6C, obtained using primers reversibly blockedat their 3′-ends, further confirmed that the next correct nucleotide,and the subsequent correct nucleotide were easily identified by usingpairwise combinations of nucleotides in the examination step. Again, thenucleotide pairs yielding the three highest magnitudes of ternarycomplex formation shared in common the presence of dTTP, therebyindicating that dTTP was the next correct nucleotide. The GT nucleotidepair yielded the highest magnitude of ternary complex formation, and soindicated the next two correct nucleotides were “TG.” FIGS. 6B-6Cgraphically present results of parameters calculated by the Octetinterferometry instrument, where the highest R_(eq) values, and thelowest k_(obs) values indicated signal strengths in the binding assay.Again, each parameter supported determination that the next two cognatenucleotides were TG.

Notably, very good sequencing results were obtained using a reiterativeprocess employing: (a) examination of a primed template nucleic acidhaving a free 3′-OH group on the primer; (b) examination with sixpairwise combinations of nucleotides, as described above; and (c)incorporation and cleavage of a reversible terminator nucleotide at the3′-end of the primer at the conclusion of the examination step. Indeed,47/47 nucleotides of a model template were determined correctly usingessentially the procedure described in Example 4, except that the Bstpolymerase was substituted in place of the Bsu polymerase. Accordingly,success of the procedure extended to multiple different DNA polymerases.

The foregoing procedures employed a plurality of examination reactionsto obtain the information needed for identifying a cognate nucleotidebefore conducting an incorporation reaction using reversible terminatornucleotides. Generally speaking, data processing for base calling neednot be contemporaneous with the examination and incorporation reactionsusing this approach. Optionally, base calling algorithms can employrecorded measurement data acquired during the examination steps.Further, it is to be understood that while examination and incorporationsteps in the illustrated protocol used two different enzymes todemonstrate procedural flexibility, these steps optionally can becarried out using the same polymerase enzyme. Still further, thereversibly blocked primer employed in the examination step permitted useof a catalytic metal ion during that step. Optionally, however,non-catalytic metal ions that inhibit polymerase-mediated incorporation,or mixtures of non-catalytic and catalytic metal ions, can besubstituted in place of the catalytic metal ions when using a primedtemplate nucleic acid molecule having a 3′-blocked primer.

Disclosed above are materials, compositions, and components that can beused for, can be used in conjunction with, can be used in preparationfor, or are products of the disclosed methods and compositions. It is tobe understood that when combinations, subsets, interactions, groups,etc. of these materials are disclosed, and that while specific referenceof each various individual and collective combinations and permutationsof these compounds may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a method is disclosedand discussed and a number of modifications that can be made to a numberof molecules including the method are discussed, each and everycombination and permutation of the method, and the modifications thatare possible are specifically contemplated unless specifically indicatedto the contrary. Likewise, any subset or combination of these is alsospecifically contemplated and disclosed. This concept applies to allaspects of this disclosure, including steps in methods using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed, it is understood that each of these additionalsteps can be performed with any specific method steps or combination ofmethod steps of the disclosed methods, and that each such combination orsubset of combinations is specifically contemplated and should beconsidered disclosed.

Publications cited herein, and the material for which they are cited,are hereby specifically incorporated by reference in their entireties.All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that the headings used herein are fororganizational purposes only and are not meant to limit the descriptionor claims.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the claims.

What is claimed is:
 1. A method of determining the identity of first andsecond correct nucleotides respectively comprising bases complementaryto the next two bases of a template strand immediately downstream of aprimer of a primed template nucleic acid molecule, said methodcomprising the steps of: (a) conducting a plurality of cycles ofcontacting a first primed template nucleic acid molecule with a reactionmixture comprising a polymerase and, for each cycle, a different testnucleotide, and removing any polymerase and nucleotide that may havebound to the first primed template nucleic acid molecule, wherein thepolymerase does not incorporate any of the different test nucleotidesinto the primer of the first primed template nucleic acid moleculeduring any of the plurality of cycles; (b) measuring, for the cycles instep (a), signals indicating the magnitudes of formation of stabilizedternary complexes comprising binding of the first primed templatenucleic acid molecule to the polymerase and the different testnucleotides; and (c) determining the identities of the first correctnucleotide downstream of the primer, and the second correct nucleotidedownstream of the primer using the measured signals from step (b),wherein the test nucleotide associated with the highest magnitude ofstabilized ternary complex formation is identified as the first correctnucleotide downstream of the primer, and the test nucleotide associatedwith the second-highest magnitude of stabilized ternary complexformation is identified as the second correct nucleotide downstream ofthe primer.
 2. The method of claim 1, wherein the primer of the firstprimed template nucleic acid molecule is an extendable primer that doesnot comprise a reversible terminator moiety attached to its 3′ terminalnucleotide.
 3. The method of claim 2, wherein the reaction mixturecomprises either a divalent non-catalytic metal ion that inhibitspolymerase-mediated incorporation, or a trivalent non-catalytic metalion that inhibits polymerase-mediated incorporation.
 4. The method ofclaim 2, wherein the plurality of cycles in step (a) comprises fourcycles.
 5. The method of claim 2, further comprising the step of (d)incorporating a reversible terminator nucleotide comprising a reversibleterminator moiety into the first primed template nucleic acid moleculeto produce a first blocked primed template nucleic acid molecule.
 6. Themethod of claim 5, further comprising the step of (e) removing thereversible terminator moiety from the first blocked primed templatenucleic acid molecule to produce a first de-blocked primed templatenucleic acid molecule.
 7. The method of claim 6, further comprisingrepeating steps (a)-(c) using the first de-blocked primed templatenucleic acid molecule in place of the first primed template nucleic acidmolecule.
 8. The method of claim 6, further comprising the steps of: (f)incorporating a reversible terminator nucleotide comprising a reversibleterminator moiety into the first de-blocked primed template nucleic acidmolecule to produce a second blocked primed template nucleic acidmolecule; and (g) removing the reversible terminator moiety from thesecond blocked primed template nucleic acid molecule to produce a secondde-blocked primed template nucleic acid molecule.
 9. The method of claim8, further comprising repeating steps (a)-(c) using the secondde-blocked primed template nucleic acid molecule in place of the firstprimed template nucleic acid molecule.
 10. The method of claim 9,wherein step (c) occurs after all of steps (a)-(b) and (d)-(g) have beenperformed.
 11. The method of claim 5, wherein none of the different testnucleotides comprises an exogenous fluorescent label.
 12. The method ofclaim 11, wherein each of the different test nucleotides is a differentnative nucleotide.
 13. The method of claim 5, wherein the polymerasedoes not comprise an exogenous fluorescent label, and wherein step (b)does not comprise measuring any fluorescent signal from the polymerase.14. The method of claim 1, wherein the first primed template nucleicacid molecule is a first blocked primed template nucleic acid molecule,and wherein the primer of the first blocked primed template nucleic acidmolecule comprises a reversible terminator moiety that blocksphosphodiester bond formation.
 15. The method of claim 14, wherein thereaction mixture comprises a catalytic metal ion.
 16. The method ofclaim 14, wherein the plurality of cycles in step (a) comprises fourcycles.
 17. The method of claim 14, further comprises the steps of: (d)removing the reversible terminator moiety from the primer of the firstblocked primed template nucleic acid molecule to produce a firstde-blocked primer; and (e) incorporating a reversible terminatornucleotide comprising a reversible terminator moiety into the firstde-blocked primer to produce a second blocked primed template nucleicacid molecule.
 18. The method of claim 17, wherein steps (a)-(c) arerepeated using the second blocked primed template nucleic acid moleculein place of the first blocked primed template nucleic acid molecule. 19.The method of claim 17, further comprising, after step (e), the steps of(f) removing the reversible terminator moiety from the second blockedprimed template nucleic acid molecule to produce a second de-blockedprimer; and (g) incorporating a reversible terminator nucleotidecomprising a reversible terminator moiety into the second de-blockedprimer to produce a third blocked primed template nucleic acid molecule.20. The method of claim 19, wherein steps (a)-(c) are repeated using thethird blocked primed template nucleic acid molecule in place of thefirst blocked primed template nucleic acid molecule.
 21. The method ofclaim 20, wherein step (c) occurs after all of steps (a)-(b) and (d)-(g)have been performed.
 22. The method of claim 17, wherein none of thedifferent test nucleotides comprises an exogenous fluorescent label. 23.The method of claim 22, wherein each of the different test nucleotidesis a different native nucleotide.
 24. The method of claim 17, whereinthe polymerase does not comprise an exogenous fluorescent label, andwherein step (b) does not comprise measuring any fluorescent signal fromthe polymerase.
 25. The method of claim 14, wherein the reversibleterminator moiety that blocks phosphodiester bond formation is a 3′-ONH₂moiety.
 26. A method of determining the identity of first and secondcorrect nucleotides respectively comprising bases complementary to thenext two bases of a template strand immediately downstream of a primerin a primed template nucleic acid molecule, said method comprising thesteps of: (a) conducting a plurality of cycles of contacting a firstprimed template nucleic acid molecule with a reaction mixture comprisinga polymerase and, for each cycle, a different pair of test nucleotides,and removing any polymerase and nucleotide that may have bound to thefirst primed template nucleic acid molecule, wherein the polymerase doesnot incorporate any of the different test nucleotides into the primer ofthe first primed template nucleic acid molecule during any of the fourcycles; (b) measuring, for the cycles in step (a), signals indicatingthe magnitudes of formation of stabilized ternary complexes comprisingbinding of the first primed template nucleic acid molecule to thepolymerase and the test nucleotides among the different pairs of testnucleotides; and (c) determining the identities of the first correctnucleotide downstream of the primer, and the second correct nucleotidedownstream of the primer using the measured signals from step (b),wherein the test nucleotide associated with the highest magnitude ofstabilized ternary complex formation is identified as the first correctnucleotide downstream of the primer, and the test nucleotide associatedwith the second-highest magnitude of stabilized ternary complexformation is identified as the second correct nucleotide downstream ofthe primer.
 27. The method of claim 26, wherein the first primedtemplate nucleic acid molecule comprises a primer with a reversibleterminator moiety that blocks phosphodiester bond formation.